Journal articles: 'UL 2 regulation'

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McCormick, Stanley R., Craig W. S. Howe, Pierre Brousset, Anil K. Tadavarthy, Cathy Quelen, Nicole Dastugue, Lisa M. Bartholomaus, et al. "Myeloproliferative Neoplasm Resembling Polycythemia Vera In a Patient with t(2;11)(p21;q23) Translocation and Persistently Elevated MiR-125b-1." Blood 116, no. 21 (November 19, 2010): 5067. http://dx.doi.org/10.1182/blood.v116.21.5067.5067.

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Abstract Abstract 5067 Background: Hematologic neoplasms associated with the chromosomal translocation t(2;11)(p21;q23) form a distinct genetic entity with diverse manifestations, and have been strongly linked with up-regulation of the microRNA miR-125b-1 (Bousquet et al, J Exp Med, 2008). Of 41 patients with myeloid malignancies and t(2;11)(p21;23) reported in the world's literature, all but 2 were cases of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), the latter cases frequently high-grade MDS transforming to AML. In 20 of 41 cases deletion of 5q was detected as a secondary cytogenetic abnormality. Only two cases of chronic myeloproliferative disorders associated with t(2;11)(p21;23) have been reported: one of chronic myeloid leukemia with a secondary t(9;22) (Ph) translocation (Royer-Pokora et al, Leukemia, 2003) and one of polycythemia vera (Acar et al, Amer J Hematol, 2006). We encountered a patient with a mixed myelodysplastic/myeloproliferative neoplasm clinically resembling polycythemia vera that was associated with t(2;11)(p21;q23) who was found to have persistent elevation in the blood of the microRNA miR-125b-1. Case Report: A 52 year-old male presented with upper extremity pain and headache. A CBC revealed the white blood cell count was 12.5 ×103/uL (55% neutrophils, 12% lymphocytes, 8% monocytes, 5% eosinophils, 21% basophils), red blood cell count 6.62 × 106/uL, hemoglobin 22.4 g/dL, hematocrit 68.1%, MCV 103 fL, and platelets 112 × 103/uL. Bone marrow aspiration and biopsy revealed a markedly hypercellular marrow (100%) with 1+ reticulin fibrosis, mild trilineal dysplastic morphology, and a blast count of 0.8%. Cytogenetic studies disclosed 46, XY, t(2;11)(p21;q23) [4]/46, idem, del (5)(q15q31) [4]/46, XY, del (5) (q13q31) [2]/46, XY[10]. FISH studies were negative for BCR/ABL1 fusion and MLL rearrangement. Molecular analysis for the JAK2V617F allele was negative. A diagnosis of mixed myelodysplastic/myeloproliferative neoplasm, unclassified was made (WHO 2008). The patient was begun on hydroxyurea, 500 mg daily. Four months after diagnosis the blood counts were within normal range, at which time quantitative real-time PCR (qRT-PCR; Bousquet et al, J Exp Med, 2008) performed on a sample of whole blood revealed a twenty-fold elevation of the microRNA species miR-125b-1, compared to healthy donors (n=3), and JAK2V617F-negative (n=3) and JAK2V617F-positive (n=2) polycythemia vera controls. Repeat qRT-PCR performed on whole blood 15 months after diagnosis confirmed persistently elevated miR-125b-1, nearly 200-fold above healthy donor controls (n=3). 18 months after diagnosis the patient remains healthy, with normal blood counts on 500 mg of hydroxyurea daily (white blood cell count 5.54 ×103/uL, red cell count 5.35 × 106/uL, hemoglobin 13.7 g/dL, hematocrit 47.2%, MCV 88.2, platelets 132.0 × 103/uL). Conclusion: This case expands the clinical-pathologic spectrum of hematologic malignancies associated with t(2;11)(p21;23), and confirms the link between t(2;11)(p21;23) and up-regulation of miR-125b-1. Translocation t(2;11)(p21;23) with miR-125b-1 up-regulation should be considered as a rare possibility in the differential diagnosis of JAK2V617F-negative polycythemia vera. Disclosures: No relevant conflicts of interest to declare.

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Gidron, Adi, John Eklund, Brenda Martone, Alfred W. Rademaker, Charles Goolsby, Laura Marszalek, and Timothy M. Kuzel. "Concurrent Treatment with Denileukin Diftitox (DD) To Deplete T-Regulatory Cells Enhances Rebound Lymphocytosis and Eosinophilia in Patients Treated with High-Dose IL-2 (HDIL-2) for Metastatic Renal Cell Cancer (MRCC)." Blood 108, no. 11 (November 16, 2006): 1729. http://dx.doi.org/10.1182/blood.v108.11.1729.1729.

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Abstract Background: CD4+CD25+hi T cells (Treg) play a suppressive role in immune regulation. DD is an IL-2 receptor specific cytotoxin. We postulated depletion of Treg with DD may enhance immune effector cell populations after HDIL-2 treatment, including rebound lymphocytosis and also eosinophilia which has been reported to be involved in immune response to neoplasm (Mattes J Exp Med 197: 387, 2003). Methods: In this pilot study, 12 pts (8 male, median age 58 yrs) with MRCC were tx with HDIL-2 and DD in different schedules to determine safety and effect on immune response as manifested by changes in Treg, peak lymphocyte, and eosinophil counts. Pts were treated with IL-2 600,000 IU/kg Q8H on days (d) 1–5 and 15–19. Three (group A) and 4 (group B) pts were given 6 and 9ug/kg daily on d8–10 respectively, while 5 (group C) pts received 9ug/kg of DD on d −4 to −2. Nine (group D) pts with metastatic melanoma who received HDIL-2 as above but without DD were included as controls. Flow cytometry was done on days −4, 1,8,10,15,22 for group C and on days 1,8,10,15,22 for groups B, and D. CBC was obtained concurrent or within 24 hours of flow cytometry. Group A pts were evaluated for safety only and were excluded from analysis. Results: Prior to enrollment, all pts had undergone nephrectomy and four patients received interferon-alpha. One pt from group B withdrew from study and was not included in analysis. Administration of DD resulted in a median decline of 25% in Treg number (not significant). DD given before HDIL-2 was associated with a greater increase in Treg post HDIL-2. In Group C there was an increase of rebound median Treg count of 0.88k/ul compared with 0.060k/ul in group B (p=0.025). Absolute lymphocytosis was higher in the combined group getting DD compared to control (median maximal increase of 7.6 vs 4.7 k/ul, respectively) although the difference did not reach statistical significance. However, group C pts had a greater increase in absolute lymphocytosis than did group B pts in which absolute lymphocytosis actually decreased (median increase 10.6 vs. median decrease 0.4 k/ul, p=0.025). A higher peak level of eosinophilia was noted in groups B and C compared with group D (mean increase of 10.5 vs. 4.0 k/ul p=0.2). Group C had a greater peak eosinophilia than group B (11.2 vs 2.2 k/ul p=0.053) Toxicity was manageable and consistent with those seen with HDIL-2. Median HDIL-2 dose given was 21 (range, 14–28). No clinical responses were observed. Of 11 pts included in the analysis 1 pt from group A expired 68 weeks after enrollment. All remaining patients are alive. Survival from enrollment ranges from 11 to 93 weeks. Conclusion: Overall, the combination of DD and HDIL-2 results in a stimulatory effect as manifested by increased rebound lymphocytosis and eosinophilia compared to HDIL-2 alone. Administration of DD in conjunction with HDIL-2 was associated with a rebound in Treg that may be schedule and dose dependent. The results suggest an enhanced immune stimulatory effect as manifested by lymphocytosis and peak eosinophilia in group C. However, this stimulatory effect also extends to Treg that may prove detrimental clinically. Further exploration of these effects in immunotherapy naïve patients would be beneficial.

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Koltsova, Alla S., Olga A. Efimova, Anna A. Pendina, Olga G. Chiryaeva, Natalia S. Osinovskaya, Natalia Y. Shved, Maria I. Yarmolinskaya, et al. "Uterine Leiomyomas with an Apparently Normal Karyotype Comprise Minor Heteroploid Subpopulations Differently Represented in vivo and in vitro." Cytogenetic and Genome Research 161, no. 1-2 (2021): 43–51. http://dx.doi.org/10.1159/000513173.

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In the present study, we aimed to check whether uterine leiomyomas (ULs) with an apparently normal karyotype in vitro comprise “hidden” cell subpopulations with numerical chromosome abnormalities (heteroploid cells). A total of 32 ULs obtained from 32 patients were analyzed in the study. Each UL was sampled for in vivo and in vitro cytogenetic studies. Karyotyping was performed on metaphase preparations from the cultured UL samples. A normal karyotype was revealed in 20 out of the 32 ULs, of which 9 were selected for further study based on the good quality of the interphase preparations. Then, using interphase FISH with centromeric DNA probes, we analyzed the copy number of chromosomes 7 and 16 in 1,000 uncultured and 1,000 cultured cells of each selected UL. All of the ULs included both disomic cells representing a predominant subpopulation and heteroploid cells reaching a maximum frequency of 21.6% (mean 9.8%) in vivo and 11.5% (mean 6.1%) in vitro. The spectrum of heteroploid cells was similar in vivo and in vitro and mostly consisted of monosomic and tetrasomic cells. However, their frequencies in the cultured samples differed from those in the uncultured ones: while the monosomic cells decreased in number, the tetrasomic cells became more numerous. The frequency of either monosomic or tetrasomic cells both in vivo and in vitro was not associated with the presence of MED12 exon 2 mutations in the tumors. Our results suggest that ULs with an apparently normal karyotype consist of both karyotypically normal and heteroploid cells, implying that the occurrence of minor cell subpopulations with numerical chromosome abnormalities may be considered a characteristic of UL tumorigenesis. Different frequencies of heteroploid cells in vivo and in vitro suggest their dependence on microenvironmental conditions, thus providing a pathway for regulation of their propagation, which may be important for the UL pathogenesis.

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Gidron, A., J. Eklund, B. Mortone, A. W. Rademaker, C. Goolsby, and T. Kuzel. "Treg depletion with denileukin difititox (DD) enhances lymphocytosis and eosinphilia in patients treated with high-dose IL-2 (HDIL-2) for metastatic renal cell cancer (MRCC)." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 14627. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.14627.

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14627 Background: CD4+CD25+ T cells (Treg) play a suppressive role in immune regulation. DD is an IL-2 receptor specific cytotoxin. We postulated depletion of Treg with DD may enhance immune effector cell populations during HDIL-2 tx, including eosinophilia which was reported to be involved in immune response to neoplasm (Mattes et al. J Exp Med 197: 387, 2003). Methods: Seven pts (5 male, median age 58 yrs) with MRCC were tx’d with HDIL-2 and DD in different schedules to determine safety and effect on immune response as manifested by changes in Treg, lymphocyte, and peak eosinophil counts. Pts were tx’d with IL-2 600,000 IU/kg Q8H on days (d) 1–5 and 15–19. Three (group A) and 2 (group B) pts were given 6 and 9 ug/kg daily on d 8–10 respectively, while 2 (group C) pts received 9 ug/kg of DD on d -4- -2. Four (group D) pts with metastatic melanoma who received HDIL-2 as above but without DD were included as controls. Flow cytometry was done on days -4, 1,8,10,15 for group C and on days 1, 8, 10, 15, 22 for groups A, B, and D. CBC was obtained within 24 hours of flow cytometry. Results: After DD Treg increased in group A (mean change in absolute T-reg count of 16%) and decreased in groups B and C (34.5 and 20% respectively) compared to baseline. Group C trended toward a greater lymphocytosis at day 8 compared to all other groups (mean increase of 8.6 vs. 3.3 K/ul p = 0.059). A higher peak level of eosinophilia was noted in group C compared with groups A, B and D combined (mean increase of 9.9 vs. 3.0 k/ul p = 0.03). Group C demonstrated a higher mean % change in absolute number of CD8+ T-cells between onset of therapy and Day 8 compared to groups A, B, D combined (increase of 1095% vs. 496% respectively, p = 0.35). Toxicity was similar to that expected with HDIL-2. Conclusions: Administration of DD in conjunction with HDIL-2 was associated with a decrease in Treg that may be schedule and dose dependent. The results suggest an enhanced immune stimulatory effect as manifested by lymphocytosis and peak eosinophilia and CD8+ T-cells in group C. Despite small pt numbers, results suggest that pre-treatment with DD may confer an advantage. It is too early to know if laboratory results correlate to clinical benefit. [Table: see text]

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Rajasekar, Reena M., Vikram Mathews, Kavitha M. Lakshmi, Auro Viswabandya, Biju George, Mammen Chandy, and Alok Srivastava. "Dendritic Cell Type 2 Counts on Day 28 in HLA-Matched Related Allogeneic PBSCT Predicts the Incidence of Acute and Chronic GVHD." Blood 108, no. 11 (November 16, 2006): 2893. http://dx.doi.org/10.1182/blood.v108.11.2893.2893.

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Abstract Dendritic cells (DC) are antigen-presenting cells involved in induction and regulation of immune responses. We investigated the impact of the number of infused and engrafted (day 28) dendritic cells, DC1 (lin−HLA-DR+CD11c+) and DC2 (lin−HLA-DR+CD123+) on the development of acute and chronic graft-versus-host disease (GVHD). Thirty three consecutive patients with hematological malignancies who underwent HLA-matched related G-CSF mobilized allogeneic PBSCT were included in the analysis. The mean follow up was 293 days (range: 36–417). There were 20 males and 13 females (median age: 29 years, range: 15–55). Conditioning regimen was myeloablative in 25 (Bu/Cy=13; Cy/TBI = 12) and non myeloablative in 8 patients (Flu/Mel = 7; Flu/Cy = 1). All patients received cyclosporine and short course methotrexate as GVHD prophylaxis. Three of them received steroids before day 28 for treatment of GVHD. Ten patients developed acute GVHD (grade II–IV) and 11 patients had chronic GVHD. The median DC2 count in the peripheral blood on day 28 was significantly lower among patients who developed acute GVHD as compared to those who did not and there was a trend to a lower count among patients with chronic GVHD [Table 1]. Based on the day 28 DC2 count patients were divided into a low DC2 quartile (<2.3cell/ul, n=8) and the rest were grouped as high DC2 (>2.3cell/ul, n=25). The two groups were comparable with regard to patient and graft characteristics (CD34, CD133, CD3, CD4, CD4CD45RO, CD4CD45RA, CD8, CD8CD45RO, CD8CD45RA, CD19, NK, DC1and DC2 cell dose). Patients in the low DC2 quartile group had higher probability of developing acute (P=0.000) and chronic GVHD (P= 0.022). Cox regression analysis revealed that low day 28 DC2 counts is associated with higher incidence of acute GVHD (HR=11.4; 95%CI=2.9–44.9; P=0.001) and chronic GVHD (HR=7.07; 95%CI=1.9–26.6; P=0.004). In a multivariate analysis which included other standard risk factors such as female to male transplants, conditioning regimen, CD34 and CD3 cell dose, low day 28 DC2 was found to be a risk factor for acute GVHD (HR=21.1; 95%CI=3–149.9; P=0.002) together with patient age (HR=1.1; 95%CI=1–1.3; P=0.039). DC1, DC2 counts in the graft and day 28 DC1 count did not correlate with the development of acute or chronic GVHD. Excluding patients who had developed acute GVHD before day 28 (n=3), a low day 28 DC2 count (<2.3/ul) had a positive predictive value of 80% for acute GVHD and 75% for chronic GVHD. These results suggest that the DC2 count in the peripheral blood on day 28 is a strong predictor for development of GVHD in recipients of PBSC in matched related allogeneic HSCT. Table 1: Median day 28 DC2 count and GVHD n, median DC2 x 103/ml (range) Yes No P-value Acute GVHD (II=IV) 10, 2.06(0.6–24.8) 23, 11.5(1.1–46.6) 0.004 Chronic GVHD 11, 2.4(1.1–46.6) 17, 8.19(1.6–44.9) 0.070

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Ohno, Tsunehisa, Mi Jin Yoo, Erik R. Swanson, Shigeru Hirano, Robert H. Ossoff, and Bernard Rousseau. "Regenerative Effects of Basic Fibroblast Growth Factor on Extracellular Matrix Production in Aged Rat Vocal Folds." Annals of Otology, Rhinology & Laryngology 118, no. 8 (August 2009): 559–64. http://dx.doi.org/10.1177/000348940911800805.

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Objectives We investigated acute changes in extracellular matrix (ECM) gene expression and histologic changes in the deposition of collagen and hyaluronan (hyaluronic acid; HA) after basic fibroblast growth factor (bFGF) treatment of the aged rat vocal fold. Methods For the polymerase chain reaction (PCR) experiments, we divided ten 18-month-old Sprague-Dawley rats into two groups that received serial injections of sham (saline solution) or bFGF (2 ng/uL) and euthanized them 2 weeks after the initial injection to investigate acute changes in ECM gene expression. We treated a separate group of 5 animals unilaterally and sacrificed them 4 weeks after the initial injection to investigate histologic changes in the deposition of collagen and HA. Results Real-time PCR revealed significantly up-regulated HA synthase (HAS)-2, HAS-3, matrix metalloproteinase (MMP)-2, and procollagen type I gene expression in the bFGF treatment group as compared to the sham treatment group. Histologic staining revealed significantly increased deposition of HA in the bFGF-treated vocal fold as compared to the sham-treated vocal fold. No differences in ECM collagen levels were observed between treatment sides. Conclusions Basic fibroblast growth factor induced the up-regulation of HAS-2, HAS-3, MMP-2, and procollagen type I. Histologically, aged vocal folds treated with bFGF revealed increased deposition of HA as compared to sham-treated vocal folds.

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Matsuoka, Ken-ichi, John Koreth, Haesook T. Kim, O. Gregory Bascug, Sean McDonough, Joseph H. Antin, Robert Soiffer, and Jerome Ritz. "Effects of Daily Low Dose IL-2 Therapy on Homeostatic Regulation of CD4+Foxp3+ Regulatory T Cells In Patients with Chronic Graft-Versus-Host Disease." Blood 116, no. 21 (November 19, 2010): 895. http://dx.doi.org/10.1182/blood.v116.21.895.895.

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Abstract Abstract 895 CD4+FoxP3+ regulatory T cells (Treg) play a central role in the maintenance of tolerance after allogeneic hematopoietic stem cell transplantation (HSCT) and recent studies have demonstrated that Treg deficiency leads to the development of chronic GHVD (cGVHD). Interleukin-2 (IL-2) is known to promote thymic generation and maintenance of peripheral Treg and IL-2 deficiency results in a profound deficiency of Treg in vivo. Based on these findings we initiated a clinical trial to evaluate the safety, clinical efficacy and immunologic effects of low dose recombinant IL-2 in patients with steroid-refractory cGVHD. We recently reported the clinical outcomes of this trial demonstrating that IL-2 administration preferentially increased Treg in patients with active cGVHD and resulted in clinical improvement with only minor toxicities (Koreth et al, ASBMT 2010). However, the mechanisms responsible for Treg expansion in patients during IL-2 administration have not been characterized. To elucidate these mechanisms, we examined phenotypic and functional characteristics of Treg in 14 patients who received daily subcutaneous IL-2 (3×105-3×106IU/m2/day) for 8 weeks. Peripheral blood samples were obtained before and at 1, 2, 4, 6, 8, 10 and 12 weeks after starting IL-2. Treg were compared to conventional CD4+FoxP3- T cells (Tcon) within individual patient samples and examined for expression of Ki-67, PD-1 and BCL-2. In some experiments, Treg and Tcon were further divided into subpopulations by the expression of CD45RA and CD31. Absolute numbers of functionally suppressive Treg increased 5-fold in the first 4 weeks of therapy. Treg numbers then slowly decreased despite continued IL-2 therapy, but remained 2-fold higher than baseline at 8 weeks. Absolute numbers of Tcon increased 2-fold in the first 4 weeks and then returned to baseline levels at 8 weeks. This resulted in a sustained increase of Treg/Tcon ratio for the entire duration of therapy, which persisted for at least 4 weeks after treatment was completed. Plasma IL-2 levels peaked at 1 week and gradually declined despite continued treatment at the same dose. Nevertheless, IL-2 levels remained significantly higher than baseline throughout treatment (median 1.4pg/ml at baseline and 18.1pg/ml at 8 weeks, p<0.05). The proliferative response to IL-2 was examined by measuring expression of Ki-67 in each subset. Initially, Ki-67 expression in Treg rapidly increased in an IL-2 dose-dependent manner. Ki-67 also increased in Tcon but at a significantly lower level (median 20.0% Treg vs 6.7% Tcon, p=0.0001). Increased Ki-67 was seen in both CD45RA+CD31+ recent thymic emigrant Treg (RTE-Treg) and CD45RA- activated/memory Treg (MEM-Treg) at similar levels (median 20.1% and 18.5%, respectively, p=0.54). Treg proliferation peaked in the first week of IL-2, and rapidly returned to baseline levels in weeks 2–3. Despite changes in proliferation, the absolute number of RTE-Treg remained significantly elevated (median 1.05/ul at baseline, 9.43/ul at 4 weeks, p=0.001). In contrast, the absolute number of RTE-Tcon did not change. Phenotypic analysis of Treg showed that expression of both PD-1 and BCL-2 increased during IL-2 therapy (%PD-1+ Treg; median 15.7% at baseline and 38.3% at 8 weeks, p=0.02: relative BCL-2 MFI; median 1.00 at baseline and 1.59 at 8 weeks, p=0.04). To determine the functional effects of these changes on susceptibility to apoptosis, Treg and Tcon were purified and cultured with or without agonistic FAS antibody, and apoptosis was measured using Annexin-V staining. Remarkably, Treg obtained during IL-2 therapy were relatively resistant to apoptosis compared to baseline. In summary, these results indicate that the selective expansion of Treg during prolonged IL-2 administration is characterized by a series of homeostatic changes. Initial high levels of IL-2 lead to selective and rapid Treg proliferation. Treg proliferation is not maintained as numbers of Treg increase and IL-2 levels decrease. Subsequent maintenance of increased Treg appears to be mediated primarily by increased resistance to apoptosis and prolonged survival. Increased thymic output of Treg also appears to support this peripheral homeostatic process. These findings demonstrate the complex effects of IL-2 on Treg homeostasis and provide important information for developing strategies to promote immune tolerance. Disclosures: No relevant conflicts of interest to declare.

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Abrahamsson, Annelie, Ifat Geron, Jason Gotlib, Jeffrey Durocher, Remi Creusot, Edward Kavalerchik, Daniel Goff, et al. "Aberrant Regulation of Wnt/Beta-Catenin Pathway Mediators in Chronic Myelogenous Leukemia Stem Cells." Blood 108, no. 11 (November 1, 2006): 2135. http://dx.doi.org/10.1182/blood.v108.11.2135.2135.

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Abstract Background Recent research suggests that self-renewing leukemia stem cells (LSC) with increased beta-catenin expression are involved in chronic myelogenous leukemia (CML) progression. We investigated whether aberrant regulation of beta-catenin destruction complex genes contributed to the enhanced self-renewal potential of CML LSC. Methods FACS Aria purified normal and CML hematopoietic stem cells (HSC), granulocyte-macrophage progenitors (GMP) and lineage positive cells were transduced for 48 hours with lentiviral luciferase GFP and transplanted intrahepatically into newborn RAG2−/−gama−/− mice. At 8 to 12 weeks human CD45+ cells were FACS-purified and transplanted into secondary recipients to establish a bioluminescent CML LSC model. RT-PCR for BCR-ABL was used to confirm CML engraftment. Wnt mediator mutation analysis was performed on cDNA via fluorescent denaturing high performance liquid chromatography (DHPLC) technology and SURVEYOR mismatch cleavage analysis both with the WAVE-HS System (Transgenomic, Gaithersberg, MD). Aliquots of PCR product (3-15 ul) from all samples were scanned for mutations by DHPLC and confirmed by Surveyor mismatch cleavage, and identified with bidirectional sequence analysis on an ABI 3100 sequencer using BigDye V3.1 terminator chemistry. Quantitative RT-PCR was also performed on CML progenitors using destruction complex gene specific primers. FACS analysis was performed with the aid of a FACS Aria to analyze changes in Wnt signaling pathway mediators. Results Advanced phase CML was typified by expansion of a GMP population with aberrantly activated beta-catenin expression, enhanced in vitro replating capacity as well as serial transplantation potential in a CML LSC bioluminescent imaging model suggesting that the GMP population was enriched for LSC (Figure 1). A targeted Wnt mutation analysis revealed a mutation in a key component of the beta-catenin destruction complex - glycogen synthase kinase 3beta (GSK) in progenitors from three of six blast crisis CML samples analyzed. Decreased GSK expression was confirmed via FACS analysis using a GSK specific antibody in three separate experiments with CML blast crisis progenitors (Figure 2). Some CML blast crisis progenitors also demonstrated a decrease in axin 2 by quantitative RT-PCR. Conclusions Altered expression of Wnt signaling pathway regulators, such as GSK3, may result in increased LSC self-renewal capacity and may represent novel therapeutic targets for advanced phase CML. Figure 1. Bioluminescent Chronic Myeloneous Leukemis stem cell Model Figure 1. Bioluminescent Chronic Myeloneous Leukemis stem cell Model Figure 2. GSK FACS Analysis. Figure 2. GSK FACS Analysis.

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Selasih, Ni Nengah. "PERANAN SENTRAL GURU AGAMA HINDU DALAM PENCAPAIAN TUJUAN PENDIDIKAN NASIONAL DI INDONESIA DAN PEMBANGUNAN KARAKTER BANGSA YANG BERAKHLAK MULIA, JUJUR, TERAMPIL, BERHATI SUCI DAN BERSIH LAHIR BATIN." Jurnal Penjaminan Mutu 1, no. 1 (February 9, 2016): 54. http://dx.doi.org/10.25078/jpm.v1i1.39.

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The teachers of Hindu religion classes play central role in the effort to reach the goal of the national education as well as to build the characters that include honesty, skillfullness, clean and good-heartedness, as described in the national standard of education regulation No 20/2003 in which it states that the curriculum has to provide religious education (Pasal 37 UU Sisdiknas). The government attention to the education is implemented too by the issue of the Regulation on Teachers and Lecturers which states that teachers are professional educators with main duty to educate, teach, guide, train, and evaluate the students in the formal elementary, secondary, and high schools (UU RI No. 14/2005) Purwanto (2004:10).Education is the intentional enlightening from the adult to the younger ones in relation to their development in order to make them useful for themselves and in the society“The Indonesian national education systemas stated in UU No.2/1989 Bab, II, pasal 4, states that the goal of the education is to develop a complete Indonesia people who are religious, good in their characters, have good knowledge and skills, healthy physically and mentally, independent, responsible for the society and nation. In line with that, the Hindu teachers should refer to the Vedic teachings and consider the physical, psychological, and social environments of study, the life as students (Sisya/Brahmacari), their roles (Acarya), the curriculum, the mteaching methods, as well as the goal of the education. These all should be centered on <ul><li>teaching with the emphasis on directing and motivating to reach the character building</li><li>facilitating that through learning experience </li><li>helping to develop attitudes, values, and self adaptation </li></ul>At schools teachers should commit themselves to be 1) role models, 2) inspirators, 3) motivators, 4) regulator, 5) evaluator besides having good vision. Without these all, the goal of education will fail.

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Geer, Eliza B., Lilian Varricchio, Fabrizio Martelli, Wu He, Lizette Couto, Yelena Lazar, Vanessa Cohen, and Anna Rita Migliaccio. "Glucocorticoid Regulation of Erythropoiesis in Humans: A Study of Patients with Cushing's Disease." Blood 126, no. 23 (December 3, 2015): 2135. http://dx.doi.org/10.1182/blood.v126.23.2135.2135.

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Abstract Cushing's disease (CD) is a rare endocrine disorder (1.2-2.4/million/year) characterized by chronic excess endogenous glucocorticoids (GC) due to an adrenocorticotropic hormone-secreting pituitary adenoma. Untreated CD results in increased mortality and multiple morbidities (obesity, diabetes, hypertension, cardiovascular disease) and, in one case report, erythrocytosis (Gursoy et al, J End Invest. 2006;29:742). The effects of chronic GC exposure on erythropoiesis in a CD cohort have not yet been studied. We prospectively quantified hematocrit (Hct), hemoglobin (Hb) and platelets (ptl) values in CD patients before (v1) and after surgical remission (v2, mean time since surgery=14.5 months) and in matched healthy controls (HC). Frequency, antigenic profiling and erythroid (Ery) expansion potential of circulating hematopoietic progenitor cells (HPC) in the three cohorts were also evaluated. The subjects analyzed included 28 v1 [6 males, 22 females, mean age=41 years; body mass index (BMI)=33.3 kg/m2 ] and 13 HC [2 males, 11 females, mean age=41 years; BMI=30.7 kg/m2 (range 26.7-34.7, p=0.073). Eleven patients were analyzed over time in both v1 and v2. Mean cortisol in v1 (22.2±6.3 ug/dL) was higher than in HC (8.5±3.8 ug/dL, p=3.4E-07) and decreased in v2 (17.6±5.1% vs. 8.7±3.9%, N= 11, p=0.0006). Hct was higher in v1 than in HC (39.8±4.7%, vs. 38.8±2.7%, p = 0.045). In the 11 patients analyzed over time, hct decreased in v2 vs. v1 (39.2±4.7% vs. 42.3±4.4%, p=0.011). Hb in v1 was not different than in HC (13.10±1.6 vs. 13.12±3.8 g/dL, p =0.225) but decreased in patients studied both in v1 and V2 (14.1±1.5 g/dL vs. 13.2±1.8 g/dL, p=0.009). Similarly, plts were not different in v1 and HC (272.3±81.4 vs. 240.8±76.0 K/uL, p=0.274) but decreased in v2 (307.7±112.1 vs. 270.6±74.5 K/uL, p=0.021). In v1, Hct did not correlate with serum (R = 0.34, p = 0.33) or 24h urine (R = 0.072, p = 0.73) cortisol concentrations. There was no difference in frequency of HPC among v1, v2 and HC [2.6±3.0, 0.34±0.28 and 1.26±0.67% of CD34+ cells and 30.2±27.2, 23.7±13.2 and 16.5±11.5 CFC/105 mononuclear cells (MNC) in v1, v2 and HC]. CD34pos cells from all groups expressed similar levels of cKIT, IL-3Rβ and prominin1, but a greater proportion of those from v1 and v2 expressed thrombospondin and thrombopoietin (Mpl) receptors than those from HC (2 vs 0.4%), suggesting that CD HPC are biased toward erythro-megakaryocytopoiesis. Consistently, in cultures without the synthetic GC dexamethasone (Dex), MNC from v1 (12±6 %) and v2 (19%) generated in 10 days a greater proportion of Erys than MNC from HC (2±1% ). However, in cultures with Dex, MNC from v1 (48±25), but not those from v2 (70±23), generated less Erys than MNC from HC (83±26 , p=0.03), suggesting that Erys from v1 HPC respond poorly to Dex. This was tested by comparing the ability of Dex to induce biochemical (GRα phosphorylation at S211 and cell-surface expression of CXCR4/calreticulin) and biological (proliferation in synergy with growth factors, GFs) responses in Erys from CD and HC. Erys from v1, v2 and HC expressed equivalent levels of GRα but those from v1 Erys contained lower levels of pGRαS211/S203 than those from v2 or HC (Fig 1A). In contrast with HC Erys, Dex decreased GRα and did not induce pGRαS211 in v1 Erys (Fig 1B). Moreover, Dex increased cell-surface expression of CXCR4 (MFI from 580 to 700) and calreticulin (MFI from 300 to 700) and proliferation (by 30%) in HC Erys but not in those from v1 s (CXCR4 MFI from 278 to 154, calreticulin MFI from 136 to 152; proliferation increases by 6%). These results indicate that chronic GC excess increases Hct values but may also activate a post-transcriptional mechanism that reduces GRα expression inducing desensitization of erythroid cells to GC. Figure 1. A) Levels of total, pS211 and pS203 GRα in Erys from HC, v1 and v2. B) levels of GRα and GRα phosphorylated at pS203 in Erys from HC and v1 exposed from 15' to Dex alone or in combination with the GR inhibitor RU486. Figure 1. A) Levels of total, pS211 and pS203 GRα in Erys from HC, v1 and v2. B) levels of GRα and GRα phosphorylated at pS203 in Erys from HC and v1 exposed from 15' to Dex alone or in combination with the GR inhibitor RU486. Disclosures No relevant conflicts of interest to declare.

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Dian Novitasari, Adinda, and Nur Samsu. "Challenges in Diagnostic and Management of Nephritic Syndrome in Diabetic Nephropathy Patient: a Case Report." Clinical and Research Journal in Internal Medicine 2, no. 1 (January 1, 2021): 158–62. http://dx.doi.org/10.21776/ub.crjim.2021.002.01.7.

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The clinical presentation of patients with acute glomerulonephritis (GN) varies widely, from asymptomatic to clinical presentations of acute kidney injury (AKI), edema, and hypertension. Diagnosis of GN in patients with diabetic nephropathy (DN) is a challenge due to pre-existing edema, hypertension, and decreased renal function. Likewise in terms of management of steroid immunosuppressants related to blood sugar regulation. It has been reported that 35-year-old male patients with diabetes mellitus (DM) with DN whose kidney function deteriorated rapidly. The patient complained of cola-red urine and decreased urine volume the day before admission. Physical examination showed blood pressure of 160/95 mmHg, bilateral leg edema, active chronic ulcer in the left lower leg, hemoglobin level was 8.7 g / dl, leukocytes 17.400 / ul, serum urea level 96 mg / dl, serum creatinine level 7.01 mg / dl (baseline level was 2.3 mg / dl), ASTO titer + 800 IU / ml, macroscopic hematuria, and albuminuria +4 on urinalysis. Ultrasonography revealed enlarged kidney size and signs of acute renal inflammation. Based on these data the patient was diagnosed clinically as rapidly progressive GN due to post-infectious GN. The patient received 3 days of pulse methyl prednisolone therapy continued orally, blood sugar regulation with insulin, RAS blockers, intravenous antibiotics and ulcer debridement. After 1 week of therapy, clinical and laboratory improvements were found and at the next followup renal function returned to baseline about 2 weeks later.

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Phillips, Stuart M., Douglas Paddon-Jones, and Donald K. Layman. "Optimizing Adult Protein Intake During Catabolic Health Conditions." Advances in Nutrition 11, no. 4 (July 1, 2020): S1058—S1069. http://dx.doi.org/10.1093/advances/nmaa047.

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ABSTRACT The DRIs define a range of acceptable dietary intakes for each nutrient. The range is defined from the minimum intake to avoid risk of inadequacy (i.e., the RDA) up to an upper limit (UL) based on a detectable risk of adverse effects. For most nutrients, the minimum RDA is based on alleviating a clear deficiency condition, whereas higher intakes are often recommended to optimize specific health outcomes. Evidence is accumulating that similar logic should be applied to dietary recommendations for protein. Although the RDA for protein of 0.8 g/kg body weight is adequate to avoid obvious inadequacies, multiple studies provide evidence that many adults may benefit from protein quantity, quality, and distribution beyond guidelines currently defined by the RDA. Further, the dietary requirement for protein is a surrogate for the constituent amino acids and, in particular, the 9 considered to be indispensable. Leucine provides an important example of an essential amino acid where the RDA of 42 mg/kg body weight is significantly less than the 100–110 mg/kg required to optimize metabolic regulation and skeletal muscle protein synthesis. This review will highlight the benefits of higher protein diets to optimize health during aging, inactivity, bed rest, or metabolic dysfunction such as type 2 diabetes.

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Wang, Chen, Weinan Wang, Lei Huang, Yoshihiro Hayashi, Xiongwei Cai, Xiaomei Yan, Gang Huang, and Yi Zheng. "RUNX1 Mutation Leads to Megakaryocyte-Primed Hematopoietic Stem Cell Expansion and Familial Platelet Disorder." Blood 136, Supplement 1 (November 5, 2020): 8–9. http://dx.doi.org/10.1182/blood-2020-140769.

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Backgrounds: Recently it is shown that there exists a subpopulation of hematopoietic stem cells (HSCs) with relatively high expression of VWF and CD41+ at the apex of the hematopoietic stem cell hierarchy. This subset of HSCs, termed mega-HSCs, can give rise to megakaryocytes and platelets directly by bypassing the traditional trajectory of megakaryocyte development from HSC via MPP and MEP. To date, aside from phenotypic marker and transplantation studies, there has been limited understanding of the mechanisms involved in the regulation of mega-HSCs. Runx1 belongs to the RUNT domain transcription factors, and is a key regulator of hematopoiesis especially for the megakaryocyte and platelet differentiation. Loss of RUNX1 in mice causes thrombocytopenia through a blockage of megakaryocyte maturation. One allele RUNX1 loss-of-function mutation is associated with familial platelet disorder (FPD) with a predisposition to developing leukemia. Here we hypothesize that Runx1 plays a role in regulating mega-HSCs to impact on platelet generation, and correcting RUNX1 mutation that causes FPD can therapeutically rescue thrombocytopenia in a mouse model. Methods: We have examined the hematopoiesis and cellularity of various bone marrow (BM) stem/progenitor populations and peripheral blood (PB) in two mouse models. Firstly, conditional knock-out of Runx1flox/flox was mediated by Mx1-Cre upon poly I:C induction. Secondly, the tetracycline-inducible RUNX1 S291fsX300 mutation was knock-in at the collagen a1 locus and the mice was crossed to MLL-PTD knock-in. The mutant RUNX1 is only expressed when the mice are fed with doxycycline and the RUNX1 mutant is "corrected" upon doxycycline withdrawn. In the second model, PB and BM were also tracked after a removal of DOX-induced RUNX1 mutation. The LSK CD150+ HSCs from the mouse BM were isolated by FACS sorting, and 10x Genomics' single-cell RNA-seq (scRNA-seq) analyses were performed to define and track HSPCs. We applied the Louvain algorithm to the scRNA-seq data to identify the cell type clusters and annotated the cell types using the marker genes of each cell cluster. MAST was employed to identify the cell-type specific, differentially expressed genes. Results: In the Runx1 KO mice, two weeks after deletion of Runx1 platelet count showed an ~2-fold decrease to 400~600 k/ul in PB. In the LSK CD150+CD48- or the LSK CD34- FLT3- compartment of BM, the CD41+ mega-HSCs increased ~2-3 fold. In the RUNX1 mutant-on model, mutant mice developed thrombocytopenia 16 weeks after DOX induction with the average platelet count dropping to ~600 k/ul and being maintained at this level. In transplant recipients, the RUNX1 mutant-on mice contained drastically increased mega-HSCs compared with RUNX1 mutant-off mice, synchronous with a decrease of the platelet count in PB. Consistent with these observations, sc-RNAseq data show that in Runx1 KO, among the sequenced LSK CD150+ cells ~70% are HSCs and ~15% are MPP2. Among the HSCs, mega-primed HSCs consist ~33% while non-mega primed HSCs are ~67%. The mega-primed HSCs are distinct by virtual of upregulated platelet-related genes and relative dormant cell cycle status. In the RUNX1 mutant-on model, we found that among the sequenced LSK CD150+ cells ~50% are HSCs and ~12% are MPP2. Interestingly, the mega-primed HSCs are significantly increased in RUNX1 mut-on HSCs compared with mut-off HSCs. Similarly, we saw highly upregulated platelet-driven pathways in mega-HSCs in the mutant-on HSCs. Finally, one month after a withdraw of DOX from the RUNX1 mutant expressing mice when the RUNX1 mutant gene became undetectable, the platelet count returned from ~600 to ~1,000 k/ul in PB, a reversal of the thrombocytopenia phenotype caused by the RUNX1 S291fsX300 expression. Importantly, the correction of Runx1 mutation significantly decreased the proportion of mega-HSCs and restored platelet related pathways in this subset of HSCs. Conclusions: Mega-HSCs contain a high level of platelet-driven gene expression. In addition to its role in regulating the HSC-MPP-MEP mediated megakaryocyte development, RUNX1 is important in regulating mega-HSCs by maintaining proper expression of mega-platelet leaning genes. Correction of RUNX1 mutation that causes FPD can rescue mega-HSC population and revert FPD, providing a rationale for future treatment strategies by gene editing in RUNX1 mutation bearing FPD patients. Disclosures No relevant conflicts of interest to declare.

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Borchers, Michael T., Nathaniel L. Harris, Scott C. Wesselkamper, Mark Vitucci, and David Cosman. "NKG2D ligands are expressed on stressed human airway epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 291, no. 2 (August 2006): L222—L231. http://dx.doi.org/10.1152/ajplung.00327.2005.

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Immune surveillance of the airways is critical to maintain the integrity and health of the lung. We have identified a family of ligands expressed on the surface of stressed airway epithelial cells whose function is to bind the NKG2D-activating receptor found on several pulmonary lymphocytes, including natural killer cells, γδ+ T cells, and CD8+ T cells. We employed real-time PCR and flow cytometry in normal and transformed airway epithelial cell to demonstrate that major histocompatibility complex class I chain-related (MIC) B and the UL-16 binding protein (ULBP) ligands (ULBP1–4) are ubiquitously expressed at the mRNA level in all cell lines. MICA/B surface expression was present on 70% of transformed cell lines but was undetectable on primary cells. We demonstrate that MICA/B and ULBP 1, 2, 3, and 4 expression is rare or absent on the cell surface of unstimulated normal human bronchial epithelial cells although transcripts and intracellular proteins are present. Normal human bronchial epithelial cells exposed to 0.3 mM hydrogen peroxide exhibit an induction of all ligands examined on the cell surface. Surface expression is independent of changes in transcript level or total cellular protein and is mediated by the ERK family of mitogen-activated protein kinases. The induction of NKG2D ligands on stressed airway epithelial cells represents a potentially important mechanism of immune cell activation in regulation of pulmonary health and disease.

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Dumitriu, Bogdan, Danielle M. Townsley, Christina Chen, Rodrigo T. Calado, Phillip Scheinberg, and Neal S. Young. "Telomere Elongation and Hematologic Improvement in Humans Treated with Androgens: A Prospective Clinical Trial of Danazol in Telomeropathies." Blood 124, no. 21 (December 6, 2014): 258. http://dx.doi.org/10.1182/blood.v124.21.258.258.

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Abstract Telomeres, the terminal complex of hexameric repeats and shelterin protein of linear chromosomes, shorten with every mitosis. Telomere attrition is accelerated in patients with mutations in telomerase complex genes (Calado and Young, NEJM 2009) and with replicative stress, as in chronic bone marrow failure. Historically, male hormones were effective in some patients with aplastic anemia (AA), and case reports and retrospective observations have suggested hematologic improvement in patients with telomeropathies treated with male hormones. Exposure of normal lymphocytes and CD34+ cells to androgens increased telomerase activity in vitro, and in cells from individuals carrying loss-of-function TERT mutations to normal levels (Calado et al. Blood 2009). We have conducted a phase I/II single-center trial (www.clinicaltrials.gov NCT01441037) assessing the safety and the effect of male hormones on telomere attrition in patients with telomere disease. Entry criteria included age-adjusted mean telomere content ≤1%ile, ± identified mutations in telomerase complex genes, and low blood counts (hemoglobin <9.5g/dL, platelets <30,000/uL, or neutrophils <1,000/uL) and/or pulmonary fibrosis. Danazol, 800 mg/day, was administered for 2 years. Primary protocol objectives were safety and activity of danazol in slowing telomere attrition. Secondary endpoints were hematologic response at 3 and 6 months (increase in hemoglobin >1.5 g/dL or platelets >20,000/uL or neutrophils >500/uL). Twenty seven patients were enrolled, accrual commencing August 2011. Most patients had moderate (n=20) or severe (n=4) AA, one had myelodysplasia, and two pulmonary fibrosis. Median age was 41 years (range 17-66); 15 patients were females. There was only one severe adverse event possibly related to drug. Frequent reported symptoms were muscle cramping with dehydration and exacerbation of headaches. Changes in serum lipid profiles were observed in all patients, with increased serum LDL and decreased HDL. Severe elevation in liver enzymes was not observed. One death occurred on study, not treatment related (pneumonia in a pulmonary fibrosis case). Mean telomere content of leukocytes at enrollment was compared with mean telomere content at 6, 12, and 24 months on drug as well as available samples before starting danazol. Telomere attrition prior to protocol entry, determined by q-pcr, was estimated at loss of 227 bp/year (95% CI, 58-368bp; p=0.009). Androgen administration appeared to elongate telomeres: the average increase in telomere length at 6 months was 205 bp (95% CI, 82-329 bp; p=0.002), at 12 months 441 bp (95% CI, 263-620 bp; p=0.0001), and at 24 months 347 bp (95% CI, 87-607 bp; p=0.01). A similar trend of increase in mean telomere content with danazol was confirmed in flow-sorted lymphocytes. Hematologic response rate, as defined by protocol, was 67% at 3 months and 60% at 6 months. Nine of eleven patients who required RBCs became transfusion-independent; two of them also became platelet transfusion independent. Liver cirrhosis was present in 6 patients at enrollment; worsening of liver disease in one occurred with continued alcohol abuse. To date 8 patients have completed two years of danazol, all of them responders; 10 patients remain on danazol, and 9 patients stopped drug prior to 2 years. Blood counts in all patients have been stable with drug discontinuation, with median follow up of 258 days (range 31-335). In conclusion, male hormones are safe and efficacious in patients with inherited bone marrow failure associated with telomeropathies, producing clinically meaningful hematologic improvement. Increased mean telomere content in patients, suggests that in vitro demonstration of up-regulation by sex hormones of TERT and of telomerase enzymatic activity is the mechanism of action of androgens in vivo. To our knowledge, this is the first successful prospective effort to favorably modulate telomere length by pharmacologic intervention in humans. Sex hormones may be useful in other conditions of accelerated telomere attrition, as for example after chemotherapy, and other drugs and small molecules may be usefully screened for their effects on telomerase in vitro. Disclosures Off Label Use: we want to determine if treatment with male hormone danazol is safe and improves hematologic response as first-line treatment in patients with AA and telomere disease(www.clinicaltrials.gov NCT01441037)..

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Liu, X., X. Liu, G. Chai, and X. Li. "SAT0263 LOW-DOSE IL-2 THERAPY EFFECTIVELY PROMOTES THE BALANCE BETWEEN TREG CELLS AND PRO-INFLAMMATORY LYMPHOCYTES IN PATIENTS WITH BEHCET’S DISEASE." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1074–75. http://dx.doi.org/10.1136/annrheumdis-2020-eular.5206.

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Background:Behcet’s disease (BD) is a chronic multisystemic disease. Several studies have shown that immune mechanisms play an important role in the development of the disease and limited options of therapeutic medicines for BD. Low dose IL-2 has been reported to selectively promote the expansion of Treg.Objectives:To evaluate the significance of Treg cells and cytokines in the pathogenesis and the changes of peripheral lymphocyte subsets and clinical indexes in patients with BD after low dose IL-2 therapy.Methods:Absolute number of CD4+CD25+FOXP3+Treg, CD4+IL17+T (Th17) and other subsets in peripheral blood (PB) from 177 patients with BD and 160 healthy donors were characterized by flow cytometry combined with an internal microsphere counting standard. And cytokines were analyzed by cytometric beads array. Thirty-nine patients were treated with daily subcutaneous injections of 0.5 million IU of human IL-2 for five consecutive days, and then its effects were analyzed.Results:As compared to healthy controls, the number of Treg cells were significantly decreased in BD patients (median:22.32 cells/ul VS median:33.12 cells/ul,P<0.001), while there was no difference about Th1, Th2 and Th17 cells. Accordingly, the median ratios of Th17/Tregs cells in patients were greatly higher than those of healthy volunteers. Besides, the circulating NK cells in the patients appeared to be lower than the proportion in the healthy donors. While no difference was observed for that of T, B, CD4+T, CD8+T cells between groups. Further, the correlation analysis showed that circulating Treg levels were negatively correlated with ESR, CRP and BDCAF respectively, suggesting an important role of Tregs in sustained high disease activity. While no obvious correlation was seen in Th1, Th2, Th17 and NK cells. Then the results showed there was a statistically significant decrease in the secretion of IL-10 in the BD patients (P= 0.004), not for IFN-γ, IL-4, IL-17 and IL-6.To evaluate the effects of IL-2 on lymphocytes in vivo, we examined 39 inpatients who received daily low-dose IL-2 at the dosage of 50 WIU for 5 days. It showed that, besides NK cells, total T cells, B cells, CD4+ T cells, CD8+ T cells, Th1 cells, Th2 cells, and Th17 cells were all increased after IL-2 treatment. But only Treg cells were amplified more dramatically, with the four-fold increase. Accordingly, the ratio of Th17/Treg was decreased significantly in patients with IL-2 treatment, tended to balance and had no difference with healthy individuals. At the same time, we found that the symptom were mitigated obviously and disease activity including ESR and CRP were both decreased distinctly without observed side effects.Conclusion:Absolute decrease of PB Tregs in patients with BD was associated with disease activity,which might be the major reason for imbalance of Th17/Tregs. It is speculated that BD is an autoimmune disease triggered by the defect of immunotolerance. More importantly, low-dose IL-2 proposes a selective biological treatment strategy by restoring immune tolerance and promoting rapidly remission.References:[1]Lopalco G, Lucherini OM, Lopalco A, et al. Cytokine signatures in mucocutaneous and ocular Behçet’s disease. Front Immunol. 2017;8:200.[2]Lucherini OM, Lopalco G, Cantarini L, et al. Critical regulation of Th17 cell differentiation by serum amyloid-A signalling in Behcet’s disease. Immunol Lett. 2018;201:38-44.[3]Noack M, Miossec P. Th17 and regulatory T cell balance in autoimmune and inflammatory diseases[J]. Autoimmun Rev,2014,13(6):668-677.Figure 1.The ratios of Th17/Treg among Healthy controls(HC), Before treatment and After treatment.Figure 2.The changes of disease activity in BD patients after low-dose IL-2 treatment.Acknowledgments: :We would like to express our sincere gratitude to all our coworkers and collaborators, to Jing Luo, Xiangcong Zhao, Chen Zhang, Qi Wu, Congcong Liang, and Rui Fu for their technical support.Disclosure of Interests:None declared

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Zhu, Yuao, Lili Huang, and David G. Anders. "Human Cytomegalovirus oriLyt Sequence Requirements." Journal of Virology 72, no. 6 (June 1, 1998): 4989–96. http://dx.doi.org/10.1128/jvi.72.6.4989-4996.1998.

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ABSTRACT The mechanisms of action and regulation of the human cytomegalovirus (HCMV) lytic-phase DNA replicator, oriLyt, which spans more than 2 kbp in a structurally complex region near the middle of the unique long region (UL), are not understood. Because oriLyt is thought to be essential for promoting initiation of lytic DNA synthesis and may participate in regulating the switch between lytic and latent phases, we undertook a mutational study to better define its sequence requirements. Kanr gene cassette insertions located an oriLyt core region between nucleotides (nt) 91751 and 93299 that is necessary but not sufficient for replicator activity in transient assays. In contrast, insertions into auxiliary regions flanking either side of this core—also required for significant replicator activity—had little effect. To search for essential components within the core region, we made a series of overlapping, roughly 200-bp deletions, and qualitatively and quantitatively assessed the abilities of the resulting constructs to mediate replication. All but one of these deletions produced a significant (i.e., greater than twofold) loss of activity, arguing that sequences across this entire region contribute to replicator function. However, two particularly critical segments separated by a dispensable region, here called essential regions I and II, were identified. Within essential region I, which overlaps the previously identified early transcript SRT, two adjacent but nonoverlapping, roughly 200-bp deletions abolished detectable replication. No single element or motif from the left half of essential region I was found to be essential. Thus, essential region I probably promotes replication through the cooperation of multiple elements. However, four small deletions in the right half of essential region I, which included or lay adjacent to the conserved 31-nt oligopyrimidine tract (referred to as the Y block), abolished or virtually abolished oriLyt activity. Together, these results identify candidate oriLyt sequences within which molecular interactions essential for initiation oforiLyt-mediated DNA synthesis are likely to occur.

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Blum, William, Rebecca B. Klisovic, Alison Walker, Ramiro Garzon, Shujun Liu, Larry J. Schaaf, Kristina Humphries, et al. "Epigenetic Targeting Via Transcriptional Inhibition of DNA Methyltransferase: a Phase I Study of Bortezomib in Combination with 5-Azacytidine in Adults with Relapsed or Refractory Acute Myeloid Leukemia (AML)." Blood 114, no. 22 (November 20, 2009): 2065. http://dx.doi.org/10.1182/blood.v114.22.2065.2065.

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Abstract Abstract 2065 Poster Board II-42 Background: Hypomethylating agents have significant clinical activity in myelodysplastic syndromes (MDS) and AML. In AML, we recently demonstrated a novel epigenetic mechanism of action for the proteasome inhibitor bortezomib (Liu, Blood 2008). Bortezomib induced hypomethylation of leukemic cells in vitro and in vivo via depletion of the Sp1/NF-kB transcriptional activation complex on the DNA methyltransferase 1 (DNMT1) gene promoter, which results in down-regulation of DNMT1 mRNA and enzyme, DNA hypomethylation and re-expression of otherwise hypermethylated target genes. Based on this preclinical work, we designed a phase I dose escalation study of 5-azacytidine (AZA) in combination with bortezomib in AML. Methods: Adults with relapsed or refractory AML by WHO criteria and preserved organ function with ECOG ≤2 were eligible. Previous decitabine or AZA was permitted. Patients received AZA at 75mg/m2 IV daily for days (d) 1-7. Bortezomib was gradually dose escalated–dose level 1 (DL 1): 0.7mg/m2 by IV push given immediately after AZA on d 2 and 5; DL 2: 0.7mg/m2 on d 2, 5, 9, and 12; DL 3: 1.0mg/m2 on d 2, 5, 9, and 12; and DL 4: 1.3mg/m2 on d 2, 5, 9, and 12. Cycles were repeated every 28 d, regardless of count recovery or response at least until 3 cycles were administered. Responses were graded by International Working Group criteria for AML (Cheson, JCO 2003). Bortezomib was discontinued after 3 cycles if no objective response of complete remission (CR), CR with incomplete count recovery (CRi), or partial remission (PR) was achieved, but AZA could be continued beyond this timepoint in the absence of disease progression. For responding patients, 12 or more cycles of therapy were permitted. Dose limiting toxicities (DLT) were assigned for cycle 1 of therapy. Given the high likelihood of infection in this population regardless of therapy, infection was not considered a DLT. Six additional patients were treated at the recommended phase 2 dose (RP2D). Results: 23 patients were enrolled with a median age of 65 years (range, 42-81) and had received a median of 2 prior inductions (range, 1-5). Median presenting WBC was 3,700/uL (500-59,100/uL); median BM blast was 26% (2-93%). 14 patients were refractory to last therapy received, including 4 with primary refractory AML. 9 patients had relapsed disease; all but 2 of these had prior CR duration <1 year. Patients received a median of 2 cycles of study therapy (range, 1-12+ cycles). Dose escalation was halted once the target bortezomib dose was reached; the RP2D was AZA at 75mg/m2 d 1-7 plus bortezomib 1.3mg/m2 d, 2, 5, 9, 12. Though no toxicities were considered to be DLT in this study, infection and/or febrile neutropenia were universal. Death within 8 weeks occurred in 5 patients (22%) due to pneumonia (1), sepsis (1), or progressive disease (3). Two patients had discontinuation of bortezomib after 2 cycles due to Grade 3 neuropathy; only 1 patient received bortezomib beyond 3 cycles. In 3 patients without objective response (and with no progression), AZA alone was continued after 3 cycles of combination therapy; each reported a subjective improvement in fatigue without bortezomib. Overall, the objective response rate was 26% (6/23). Responses were as follows: 3- CR, 2- CRi, and 1-PR. One CRi patient (in cytogenetic remission also) who discontinued study treatment after 2 cycles due to unrelated trauma subsequently had complete count recovery, but a repeat marrow examination was not performed. Three patients went on to allogeneic transplantation due to response achieved. Response followed the typical pattern of azanucleoside activity, requiring more than one cycle of therapy; the median number of cycles to initial response was 2 (range, 1-5). 5/6 responders had response to combination therapy; one patient responded following 5 cycles of treatment, the last 2 cycles with AZA as a single agent. Conclusions: The combination of 5-azacytidine and bortezomib is well tolerated and active in this cohort of relapsed or refractory AML patients. Additional studies to further elucidate the role of proteasome inhibition as a mediator of hypomethylating activity in AML are warranted. Correlatives studies are ongoing. Disclosures: Blum: Celgene: Research Funding.

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Diarra, A., N. Eissa, and J. Ghia. "A212 CHROMOFUNGIN PROTECTS AGAINST DSS-INDUCED COLITIS BY REGULATING P-53 APOPTITIC PATHWAY." Journal of the Canadian Association of Gastroenterology 3, Supplement_1 (February 2020): 84–85. http://dx.doi.org/10.1093/jcag/gwz047.211.

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Abstract Background Development of ulcerative colitis is associated with epithelial apoptosis mediated by p53-apoptotic pathway through the activation B-cell lymphoma 2 (Bcl-2), Bcl-2 associated-X protein (BAX) and Bcl-2 antagonist/killer-1 (BAK1) proteins. Chromofungin (CHR), a chromogranin-A derived peptide expressing a cell penetrating peptide motif, decreased the severity of colitis via the suppression of mucosal and pro-inflammatory macrophages-related p53-dependent apoptosis. Aims We aimed to investigate a) whether the gene profile expression of apoptosis could be extended to other p53-associated genes; b) whether the gene expression of some of the p53-apoptosis marker could be confirmed by protein analysis; and c) whether due to the cell penetrating peptide motif, CHR could enter into peritoneal macrophages. Methods UC-related colitis was induced in C57BL/6 mice (7 weeks) by administering dextran sulfate sodium (DSS) (5%, 5 days). Preventive CHR (2.5 mg/kg/day) or vehicle treatment started 1-day before colitis induction and lasted for 5-days. Profiler™ PCR Array was performed to screen a panel of 84 genes representative of the p53 signal pathway in colitic whole mucosa distal colonic samples treated or not with CHR. Western blot analysis was performed to confirm individual protein changes. Naïve macrophages were plated overnight and nonadherent cells were removed the next day. Cells were incubated with rhodamined CHR (4 ul) for 5, 10, 20, 30 min before washing and fixing them, detection was made via confocal microscopy. Results In colitic conditions, an up regulation of 26 genes associated to the p53-dependent apoptosis pathway were detected including Apaf1, Bax, Bbc3, Bcl2, Cradd, Fadd, Cul9, Pmaip1, Tnfrsf10b. In vivo CHR treatment decreased significantly the colitis and was associated with a significant downregulation of 19 genes including the 9 aforementioned when compared with biopsies from colitic groups. Compared to untreated groups, colitic mice treated with CHR demonstrated a significant decrease of BAX and BAK protein and the apoptotic ER stress inducer marker, X-Binding Protein 1. A large number of peritoneal macrophages displayed rhodamine within the all intracellular compartment. The presence of the peptide inside the cell can be visible as early as 5 min and the signal gradually increases. Conclusions CHR decreases the inflammatory process via the suppression of a large number of p53-related apoptotic proteins. CHR quickly enters the macrophage but the exact mechanism of entrance needs to be further defined. Targeting functional analysis of CHR may lead in the future to novel therapeutics for UC. Funding Agencies CCCNSERC

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Amaru, Ricardo, Ariel Amaru, Hortensia Miguez, Torres Gina, Josue Mamani, Oscar Vera, Heriberto Cuevas, Josef T. Prchal, and Jaroslav F. Prchal. "Bolivian Aymara Natives with Chronic Mountain Sickness Have Autonomous BFU-E Growth." Blood 126, no. 23 (December 3, 2015): 5206. http://dx.doi.org/10.1182/blood.v126.23.5206.5206.

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Abstract Background Erythrocytosis / polycythemia is divided into primary and secondary. Primary polycythemia can be either acquired; i.e. polycythemia vera (PV) due to somatic JAK2 mutation, or congenital due to germ-line DNA changes (erythropoietin (EPO) receptor and VHL mutations in Chuvash polycythemia). These mutations are expressed within erythroid progenitors, drive increased erythropoiesis and are detected by hypersensitive or autonomous EPO BFU-E responses. In contrast, secondary erythrocytosis (SE), such as seen with cardiopulmonary pathologies, is driven by the circulating EPO. Chronic mountain sickness (CMS) is characterized by high altitude pathological erythrocytosis and by cognitive and neurological impairments. CMS is found in subjects living in high altitude (2500 meters and higher). In La Paz, Bolivia, (3600m) there is 7% incidence of CMS erythrocytosis. Some human populations (Tibetans, Andean Quechuas and Aymaras, and Ethiopians) are adapted to very high altitudes and their adapted phenotypes and, in some instances, evolutionarily selected haplotypes, have been reported. Whole genome was evaluated in Andeans and two genes, SENP1 and ANP32D were found to be evolutionarily selected and correlated with presence or absence of erythrocytosis. The genes down-regulation in hypoxia had survival benefit in Drosophila ortholog (1).SENP1 desumoylate GATA-1 and other regulatory proteins and is critical for definitive erythropoiesis (2,3). Here we evaluated native Aymara La Paz dwellers with three types of polycythemia: CMS, SE secondary to cardiopulmonary disease, and PV, by clinical studies and by in vitro evaluation of erythroid progenitors, and compared them to non-polycythemic subjects. Patients and Methods Complete blood count was performed by automatic hematologic counter (Micro 60, USA). Serum EPO was measured by Elisa (R&D System, USA) and JAK2V617F mutation analysis by PCR assay. Erythroid progenitors were isolated by density gradient centrifugation and cultured in methylcellulose medium with and without EPO (Stem Cell technologies, Canada) at 370 C and 5 % CO2. BFU-E colonies reading was carried out according to standardized criteria at 7 and 14 days. Results Table. Normal Control(n=10) CMS (n=15) Secondary Erythrocytosis(n=10) PolycythemiaVera (n=5) 1.Gender M/F 10/0 15/0 10/0 3/2 Age (range) 42 (40-47) 48 (29-58) 53 (34-72) 67 (42-74) Hb g/dl (SD) 16.2 (+ 0.9) 20.3 (+ 0.9) 22.8 (+ 1.4) 20.0 (+ 2.5) Ret % (SD) 1.3 (+ 0.1) 2.9 (+ 1.3) 3.6 (+ 1.2) 2.1 (+ 0.3) WBC /ul (SD) 6300 (+ 1600) 7200 (+ 1900) 6600 (+ 1700) 16600 (+ 4800) PLT 103 ul (SD) 273 (+ 80) 229 (+ 58) 193 (+ 54) 604 (+ 177) sEPO mUI/ml (SD) 10.0 (+ 3.9) 10.5 (+ 2.2) 82.9 (+ 30.4) 3.0 (+ 1.2) JAK2 V617F, No. (%) 0 (0) 0 (0) 0 (0) 100 Apoptosis Normal Delayed Normal Delayed BFU-E: EEC 0 (0-0) 10 (2-25) 0 (0-0) 45 (25-70) References: 1. Yu L et al. J Exp Med., 2010, 207:1183. 2. Sharma D et al. Cell Report, 2013, 3:1640. 3. Zhou D et al. Am J Hum Genet. 2013, 93:452. 4. Kapralova K et al. Blood. 2014,123:391 Conclusions a) Endogenous erythroid colony (EEC) are present in Aymaras with CMS, indicating primary polycythemia. b) Endogenous EECs are higher in PV than in CMS. c) CMS subjects have normal serum EPO levels. d) The role of SENP1, and hypoxia-regulated RUNX1 and NF-E2 (4) that promote erythropoiesis, is being interrogated in native erythroid cells. e) It remains to be determined if the autonomous BFU-E growth is specific for Aymara's CMS or present in CMS individuals of other ethnicities. Disclosures No relevant conflicts of interest to declare.

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Badar, Talha, Hagop M. Kantarjian, Susan O'Brien, Guillermo Garcia-Manero, Elias Jabbour, Rebecca Garris, Naveen Pemmaraju, et al. "Clinical Outcome of De Novo Adult Acute Lymphoblastic Leukemia (ALL) with 11q23/Mixed Lineage Leukemia (MLL) Gene Rearrangements." Blood 124, no. 21 (December 6, 2014): 5342. http://dx.doi.org/10.1182/blood.v124.21.5342.5342.

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Abstract Introduction: The MLL gene on located on 11q23 plays an essential role in positive regulation of gene expression in early embryonic development and hematopoiesis. Fusion genes, such as MLL-AF4 resulting from t(4:11)(q21;q23), alter normal cellular proliferation and differentiation, favoring leukemogenesis. The DOT1L histone methyltransferase associated with MLL appears to be required for maintenance of ALL. MLL-AF4 is detected in about 10% of de novo B-lymphoblastic leukemia or 30=40% of pro-B ALL subtypes. The 11q23/MLL subtype of ALL has been associated with extremely poor outcomes. We conducted a retrospective analysis of de novo adult ALL with 11q23/MLL gene rearrangements. Methods: Overall the database included 74 pts with 11q23/MLL gene rearrangements referred to our institution between 1980 and 2014. Of these, 20 (27%) pts were relapsed/refractory, 4 (5%) were minimally pretreated, 5 (7%) were inevaluable for response, 3 (4%) had concomitant Philadelphia chromosome, 2 (3%) were T-lineage, 2 (3%) were mature B-ALL. The remaining 38 cases comprised the study cohort evaluated with respect to pretreatment characteristics and outcome measures such as response to therapy, remission duration (CRD), and survival (OS). Results: Baseline characteristics of the cohort included median age 44 yrs (range, 20-75); 26% were older than 60 yrs. Ten (26%) pts had a prior malignancy and were designated secondary ALL. Five (13%) pts had CNS disease at presentation. Median WBC count was 69.8 K/uL (range, 0.5-612); 24 (63%) pts had WBC > 30 K/uL. Nineteen (50%) pts had serum LDH > 1400 U/L. Co-expression of myeloid markers (CD13, CD33 +/- CD117) was noted in 6 (16%) pts. Distribution of the 11q23/MLL gene aberrancies were: t(4;11) (q21; q23) in 25 (66%) pts, 11q23 without translocation partner in 10 (26%) pts, and t(11;19)(q23;p13.1) in 3 (8%) pts. Thirty two (84%) pts were treated per the hyper-CVAD (cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with high dose methotrexate and cytarabine) regimen (3 [10%] received concurrent rituximab) and 6 (16%) pts were treated per the augmented BFM regimen. Overall response rate was 95%; complete remission (CR) in 92% and CR without platelet recovery (CRp) in 3%. Overall median CRD was 26.3 mos (range 0.2-93+ mos); median OS was 24.2 mos. Overall, the 5-yr CRD and OS rates were 19% and 26%, respectively, with a median follow-up of 60 mos (range, 4.8-173+ mos). Median OS in pts with 11q23 without chromosomal translocation was 48.5 mos, compared with 24 mos for t(11; 19) and 13.3 mos for t(4; 11) (p= 0.07) (Fig. 1). There were no differences in outcomes by older age or designation as secondary ALL. Conclusion: The 11q23/MLL positive B-lymphoblastic subsets are associated with differentially inferior outcomes, although MLL-AF4 remains the ALL subset associated with highest risk of disease recurrence particularly in the absence of allogeneic stem cell transplantation; the relatively rarity of these subsets poses challenges in establishing the optimal treatment strategies. Therapeutic approaches incorporating hypomethylating agents, histone deacetylase inhibitors, and CAR-T cells could improve these dismal outcomes; targeted agents such as DOT1L inhibitors are under investigation in clinical trials. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Saida, Satoshi, Tao Zhen, Erika Mijin Kwon, Guadalupe Lopez, and Paul P. Liu. "Distinct Roles of GATA2 in Development and Evolution of CBFB-MYH11 AML." Blood 132, Supplement 1 (November 29, 2018): 770. http://dx.doi.org/10.1182/blood-2018-99-110636.

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Abstract Introduction. Core binding factor acute myeloid leukemia (CBF-AML) is caused by the dysfunction of a heterodimeric protein complex composed of the transcription factor RUNX1 and its partner CBFb. An inversion of chromosome 16 generates a fusion between CBFB and MYH11. The encoded fusion protein, CBFb-smooth muscle myosin heavy chain (SMMHC), contributes to the pathogenesis of CBF-AML. Our previous reports suggest that CBFB-MYH11 contributes to leukemogenesis by up-regulation of genes such as Gata2, which is an essential hematopoietic transcription factor. On the other hand, we recently identified recurrent monoallelic deletions of GATA2 on chromosome 3 in relapsed CBF-AML patients (Sood et al., Leukemia 30:501-504, 2016). From these findings we propose two hypotheses; 1) up-regulation of GATA2 contributes to leukemogenesis by CBFB-MYH11 in the initiation phase; 2) GATA2 deficiency contributes to the relapse of CBF-AML. Methods. Two datasets (GSE19194 and GSE102388) from microarray and RNA-Seq were used to determine Gata2 expression level in Cbfb-MYH11 preleukemic murine hematopoietic cells. Cbfb-MYH11 conditional knock-in (Cbfb+/56M), Gata2 conditional knockout (Gata2+/f), and Mx1-Cre transgenic mice were crossed to generate Gata2+/fCbfb+/56MMx1-Cre mice. Mice were injected with pIpC to induce the expression of Cbfb-MYH11 and/or knockout of Gata2 through Cre-recombinase activation. For transplantation assays, spleen cells obtained from leukemic mice were injected into irradiated recipient mice through tail vein. For in vitro colony forming assays, colonies were counted after 10 days in culture. Cell apoptosis was determined by Annexin V and 7AAD staining. Results. To test the first hypothesis, we determined the expression level of Gata2 in preleukemic cells in the Cbfb-MYH11 expressing mice. Data from both microarray and RNA-seq experiments revealed that Gata2 was highly expressed in the preleukemic hematopoietic cells of the Cbfb-MYH11 mice, as compared to those of the WT mice, and this finding was confirmed by qRT-PCR. Based on published ChIP-seq data, Gata2 is likely a direct transcriptional target of CBFb-SMMHC. Next, we determined the impact of Gata2 deficiency on leukemogenesis by Cbfb-MYH11. qRT-PCR showed reduced Gata2 expression in bone marrow cells from Gata2+/fCbfb+/56MMx1-Cre mice 12 days after pIpC injection (0.029±0.0092 vs 0.076±0.014; p=0.0089). Colony forming ability was decreased for the pre-leukemic bone marrow cells in Gata2+/fCbfb+/56MMx1-Cre mice when compared to Cbfb+/56MMx1-Cre mice (mean 37.2±6.35 vs. 74.23±8.335; p=0.0002). In addition, the Gata2+/fCbfb+/56MMx1-Cre mice had a smaller abnormal myeloid population in the bone marrow, which is capable of inducing leukemia, when compared with Cbfb+/56MMx1-Cre mice (mean 0.43±0.14% vs. 1.42±0.34%; p=0.0092). Most significantly, Gata2+/fCbfb+/56MMx1-Cre mice developed leukemia with a much longer latency than Cbfb+/56MMx1-Cre mice (median survival 215 days vs 125 days; p=0.0007). To test hypothesis 2, we compared the phenotype of the end stage mice for each genotype. Gata2+/fCbfb+/56MMx1-Cre mice had higher WBC count in peripheral blood than Cbfb+/56MMx1-Cre mice (mean 92,000±20,429 cells/ul vs. 35,644±12,001 cells/ul; p=0.0243), which is a poor prognostic marker in human leukemia. Leukemic cells from Gata2+/fCbfb+/56MMx1-Cre mice also had lower percentage of Annexin V positive cells than Cbfb+/56MMx1-Cre mice in short term culture (31.0±7.1 vs. 68.9±6.5%; p=0.0117). More importantly, upon transplantation, the recipient mice transplanted with Gata2+/fCbfb+/56MMx1-Cre leukemia cells developed leukemia much faster than recipient mice transplanted with equal numbers of Cbfb+/56MMx1-Cre leukemia cells (median survival 35.5 vs. 91.0 days; p<0.0001). Conclusions. Our findings suggest that Gata2 plays important but distinct roles in two different stages of Cbfb-MYH11 leukemia. Reduction of Gata2 activity delays leukemia development in primary Cbfb-MYH11 knockin mice, while contributing to a more aggressive phenotype in leukemic phase as shown in primary leukemic mice and transplanted recipients, which may be correlated with leukemia relapse in human patients. We are analyzing data from whole exome sequencing and RNA-seq to understand the mechanism underlying the observed phenotypes, and the findings will be presented at the annual meeting. Disclosures No relevant conflicts of interest to declare.

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Blum, William, Rebecca B. Klisovic, Ramiro Garzon, Alison Walker, Sebastian Schwind, Shujun Liu, Larry Schaaf, et al. "Phase 1 Trial of Decitabine and Bortezomib in High Risk Acute Myeloid Leukemia (AML)." Blood 116, no. 21 (November 19, 2010): 3293. http://dx.doi.org/10.1182/blood.v116.21.3293.3293.

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Abstract Abstract 3293 Background: The hypomethylating agent decitabine has significant activity in AML. We previously demonstrated a novel epigenetic mechanism of action for the proteasome inhibitor bortezomib in AML cells (Liu, Blood 2008). Bortezomib induced hypomethylation of leukemic cells in vitro and in vivo via disruption of the Sp1/NF-kB transcriptional activation complex on the DNA methyltransferase 1 (DNMT1) gene promoter, which resulted in down-regulation of DNMT1 mRNA and protein levels, DNA hypomethylation, and re-expression of otherwise hypermethylated target genes. Based on this, we designed a phase 1 dose escalation trial of decitabine in combination with bortezomib. Methods: Adults with high risk AML who had preserved organ function and ECOG ≤2 were eligible. High risk AML included relapsed/refractory AML or age>60 years with previously untreated disease (if ineligible for or refused standard induction therapy). Patients received decitabine at 20mg/m2 IV daily for days (d) 1–10 of 28 d cycles with dose modification in subsequent cycles based on response and myelosuppression. Bortezomib (given immediately after decitabine) was gradually dose escalated in standard 3+3 fashion from 0.7mg/m2 on d 5 and 8 to the target dose of 1.3mg/m2 on d 5, 8, 12, and 15. Cycles were repeated every 28 d, regardless of count recovery. The plan was to administer 3 cycles if possible before discontinuation due to lack of response. For responding pts, therapy was continued indefinitely. Six additional pts were treated at the recommended phase 2 dose. Dose limiting toxicities (DLT) were assigned for cycle 1 of therapy. Given the high likelihood of infection in this population regardless of therapy, infection was not considered a DLT unless toxicity exceeded that expected with conventional therapy. Results: 19 pts were enrolled with a median age of 69 years (range, 32–83). 12 pts were age>70. 10 pts were previously untreated. Median presenting WBC count was 3,900/uL (range, 1,300-69,200/uL); median bone marrow blast was 34%. Patients received a median of 2 cycles of therapy (range, 1–14 cycles). Two pts received combination therapy beyond 3 cycles of treatment; one received combination for 8 cycles, then decitabine alone for 6 additional cycles, and the other received combination for 4 cycles, then decitabine alone for 6 additional cycles. Dose escalation was halted once the target bortezomib dose was reached; the MTD was decitabine at 20mg/m2 d 1–10 plus bortezomib 1.3mg/m2 d 5, 8, 12, and 15. One DLT of death due to sepsis occurred in dose level 3. Febrile neutropenia and infectious complications were frequent. Death within 8 weeks occurred in 4 pts (21%). Neuropathy attributable to bortezomib, though not meeting DLT criteria, was problematic with repetitive cycles of administration. Specifically, two pts had Grade 3 neuropathy requiring discontinuation of treatment. Responses occurred in 4/10 previously untreated pts: 3 had complete remission (CR) and one had CR with incomplete blood count recovery (CRi). One 84 year old pt with complex karyotype discontinued therapy after one cycle due to fatigue with persistent disease (non-responder). Notably, the pt subsequently had complete count recovery and lived for 14 months with no additional treatment, refusing further marrow evaluation of response. Response durations for the 3 CR were as follows: 12 months, 9 months, and the last patient died in CR after 10 months response duration due to unrelated and preexisting cardiac disease. The CRi lasted 3 months before relapse. Responses occurred in 2/9 relapsed/refractory pts. Both had CRi. Response duration for one patient was only 2 months. The other had allogeneic transplant in first remission 22 months ago before recently expiring in remission with transplant-related complications. In 3/6 responders with abnormal karyotype, two achieved cytogenetic CR. Conclusions: The combination of decitabine and bortezomib was reasonably well tolerated and active in high risk AML. Neuropathy beyond cycle 1 limited prolonged exposure to both agents. Given recent data suggesting equivalent efficacy with weekly dosing of bortezomib in multidrug treatments for myeloma, modification of the bortezomib schedule may facilitate more prolonged exposure to the combination. A phase 2 study of this combination is being planned, with bortezomib modification as noted. Correlative studies are ongoing. NCI U01 CA 76576, NIH/NCI K23CA120708. Disclosures: Blum: Celgene: Research Funding. Off Label Use: decitabine and bortezomib in AML.

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Reading, N. Scott, Archana M. Agarwal, Ronald Hoffman, Josef T. Prchal, and Mohamed E. Salama. "Transcriptional Characterization of Myelofibrotic Bone Marrow Microenvironment Reveals Distinct Tumor Microenvironment in JAK2+ and Calr+ PMF Marrows." Blood 128, no. 22 (December 2, 2016): 1954. http://dx.doi.org/10.1182/blood.v128.22.1954.1954.

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Abstract Background: Primary myelofibrosis (PMF) is a clonal stem cell disorder associated with somatic mutations in three genes: Janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL). Although, our understanding of the microenvironment in PMF is limited, in PMF levels of Treg, cytotoxic T-cells, B-cells, macrophages and megakaryocyte cell populations have been reported to be elevated in either peripheral blood or bone marrow (BM) (Barosi Curr Hematol Malig Rep 2014). In addition, various cellular pathways including JAK/STAT, TGFβ1, and cytokine pathways (CXC family, hematopoietin family, PDGF family and TGF family), have been reported to play an important role in the dysregulation of hematopoietic cell proliferation and disease progression. Here-in we characterize the tumor microenvironment in formalin fixed paraffin embedded (FFPE) BM biopsies obtained from PMF patients and correlate these findings with mutational status. Methods: We applied the enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) to identify and assess immunological function in the microenvironement of archival FFPE bone marrow samples from patients with PMF. Twelve archival bone marrow FFPE biopsies from PMF patients along with clinical information and 5 normal controls were analyzed using upto 500ng of RNA (at 100ng/ul) for digital expression profiling. The panel included 109 genes that define 24 immune cell types and populations and forty housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 3.0 and the Advanced Analysis Module (v.1.0.84). Results: Gene expression profiles for cellular immune pathways were analyzed for global changes based mutation. Globally, cellular functions involving immune cell development and cellular responses/functions were dramatically decreased in myelofibrotic marrow (chemokines, complement, cytokines, cytotoxicity) when compared to normal marrow. However, only in areas of adhesion, antigen processing, transporter function and senescence genes were transcription levels elevated over normal controls. Differential expression analysis of JAK2V617F+ marrow showed decreased expression of genes involved in cell regulation, NK cell function, T-cell functions and pathogen defense and increased expression of genes involved in inflammation, chemokines and transporter functions over normal marrow. Whereas CALR+ bone marrow biopsies showed fewer genes down regulated and an increased number of genes up regulated, particularly involved in fibrosis, inflammation, chemokines, adhesion, antigen processing and regulation. Pathway analysis suggested a particular role for FLT3 ligand in myeloid stem cell regulation, thrombospondin (THBS1) which has been reported to promote the activation of the latent forms of TGFβ1, and mitogen-activated protein kinases (JNK1, ERK) in PMF cell proliferation and differentiation. Conclusions: Digital immune expression profiling reveals a distinct PMF tumor microenvironment and illustrates potential transcriptional differences based on their mutational status. (JAK2+ or CALR+). These transcriptional changes in myelofibrotic marrow are reflected in global changes in immune cells and pathway activation These data provide for the first time in situ evidence of the importance of the immune system in PMF pathogenesis. Barosi G, 2014 An immune dysregulation in MPN. Curr Hematol Malig Rep 9:331-339. Disclosures No relevant conflicts of interest to declare.

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Ramos, Pedro, Sara Gardenghi, Robert W. Grady, Maria de Sousa, and Stefano Rivella. "ß-Thalassemic Mice Require Functional Hfe to Modulate Hepcidin Expression In Response to Iron Overload." Blood 116, no. 21 (November 19, 2010): 2063. http://dx.doi.org/10.1182/blood.v116.21.2063.2063.

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Abstract Abstract 2063 ß-Thalassemia is a genetic disorder characterized by decreased or absent production of ß-globin chains, leading to ineffective erythropoiesis, anemia and iron overload. Hepcidin, the hormone that controls iron homeostasis, is regulated by several mechanisms, including erythropoiesis, iron overload, inflammation and hypoxia. In the absence of transfusion therapy, patients with ß-thalassemia major exhibit a severe ineffective erythropoiesis that suppresses hepcidin expression. However, in patients or animal affected by ß-thalassemia intermedia (th3/+), iron overload is associated with a milder form of ineffective erythropoiesis. In this study we investigated whether th3/+ mice retain the ability to modulate hepcidin expression in response to iron load, despite their increased erythropoietic activity. We analyzed some of the genes involved in the regulation of hepcidin, in particular, genes that are upregulated by iron overload in wt mice. These included Bmp6, a strong modulator of Hamp in response to iron, and Id1, Atoh8 and Smad7, other targets of the Bmp/Smad pathway. Analysis of the phosphorylation of the Smad protein complex is in progress. In addition, we generated mice affected by ß-thalassemia intermedia lacking the Hfe gene (Hfe-th3/+), in an attempt to determine whether or not this gene is involved in hepcidin regulation in this disorder. We analyzed th3/+ mice at 2, 5 and 12 months of age. In 2-month-old th3/+ mice hepcidin expression was significantly low compared to wt mice. As th3/+ mice age and their iron overload worsens, hepcidin expression increases showing similar and elevated levels in th3/+ compared to wt animals, respectively at 5 and 12 months. At 2 months, hepcidin expression normalized to liver iron concentration exhibited even lower levels in th3/+ mice compared to wt animals. This ratio did not change in aging th3/+ animals, despite the fact that their liver iron concentration increased over time (0.66, 1.24, and 1.45 ug/mg of dry weight at 2, 5 and 12 months, respectively). The expression levels of Bmp6, Id1, Atoh8 and Smad7 followed a similar pattern, being generally downregulated at 2 months compared to wt mice. However, as iron overload progressed, th3/+ mice exhibited increased expression of these genes compared to wt mice. Similar to what was observed with hepcidin, their expression was low in th3/+ mice at all ages when normalized to liver iron concentration. These observations indicate that hepcidin expression in ß-thalassemia increases over time and is regulated by the relative levels of ineffective erythropoiesis and iron overload. We also investigated the relationship between Hfe and hepcidin in response to iron in ß-thalassemia. We transplanted the ß-thalassemic phenotype into lethally irradiated wt or Hfe-KO mice, generating th3/+ and Hfe-th3/+ animals, respectively. Compared to th3/+ mice, we observed that Hfe-th3/+ animals had increased hepatic iron (3.09 vs 1.29 ug/mg of dry weight, p≤0.05) and serum iron (232 vs 162 ug/dL, p≤0.05), with no significant changes in splenic iron concentration. The Hfe-th3/+ mice also exhibited increased hemoglobin levels (9.4 vs 7.8 g/dL, p≤0.001) due to an increase in both red cell counts (8.9 vs 8.0 ×106 cells/uL, p≤0.01) and mean corpuscular hemoglobin levels (10.6 vs 9.7 pg, **p≤0.05). However, this did not reduce splenomegaly or ineffective erythropoiesis. We also analyzed the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 in 5-month-old mice. At his time point expression of most of these genes was similar between wt, th3/+ and Hfe-th3/+ mice. Only expression of Bmp6 was elevated in the two thalassemic groups compared to wt mice. When the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 expression were normalized to liver iron content, we observed significant reductions in Hfe-th3/+ mice compared to th3/+ animals. Taken together, these observations indicate that iron overload can partially counteract the repressive effect of ineffective erythropoiesis on hepcidin expression in th3/+ mice. Moreover, lack of Hfe further impairs the ability of hepcidin and other iron regulated genes to respond to iron overload, aggravating this feature in thalassemic mice. Overall, this indicates that Hfe plays a positive role in the regulation of hepcidin in ß-thalassemia. Disclosures: No relevant conflicts of interest to declare.

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De Tullio, Giacoma, Carla Minoia, Margherita Gigante, Sabino Strippoli, Simona Serratì, Giacomo Loseto, Rosa Angarano, et al. "Relevance of αβ-DNTs as Prognostic Factor on Clinical Outcome and Role on Tumor Surveillance in Haematological Diseases and Solid Tumor: Preliminary Evaluations." Blood 124, no. 21 (December 6, 2014): 3840. http://dx.doi.org/10.1182/blood.v124.21.3840.3840.

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Abstract The mutual interactions between the host immune system and cancer cells may either promote or control oncogenesis. Cancer immunotherapies remain viable approaches to sustain patient survival. However, positive clinical response of phase I/II clinical trials remains still to low. The Double negative T cells (DNTs) have emerged as new subset of T cells that contributes specifically to anti- tumor immunity since involved in immune regulation and tolerance acting as regulatory T cells (Treg) and/or cytotoxic T cells. DNTs express either αβ or γδ T-cell receptors (TCR) or lack CD4, CD8 and CD56. Functional studies showed that DNTs might have a direct in vitro anti-tumor activity against lymphoma and melanoma cells. However no data are available on their prognostic significance, modulation during the therapy response and their ability to kill tumor cells in cooperation with other immune cells such as DCs to explain their role in human anti-lymphoma immunity. Knowledge of the DNTs role in tumor surveillance and as prognostic factor for clinical outcome has a strong clinical valence and might allow us to design new approaches of therapeutic strategy. The aim of this study was to evaluate the DNTs during therapy response in lymph nodes (LN), bone marrow (BM) and peripheral blood (PB) of both lymphoma (Ly), Renal Cell Carcinoma (RCC) and Metastatic Melanoma (MM) patients (pts) in order to assess their role on clinical outcome, progression and tumor surveillance. PB and BM samples of 90 Ly pts, with NHL and cHL were collected at diagnosis and during the follow-up (1-6-12 months after chemo- or immuno-chemotherapy therapy). PB samples of 16 healthy donors were collected as control. Twenty fresh LN tissues from pts clinically suggestive of Ly were quickly processed. To evaluate the interaction of DNTs with tumor burden either 22 samples from RCC pts and 30 samples from MM pts were collected. The ontogeny, functional attitude and TCR clonality of DNT subsets (TCRαβ+ and TCRγδ+) were evaluated by following antibodies: CD3, CD4, CD8, CD56, CD45, TCRαβ, CD45Ra, CD45Ro, CCR7, CD27, CD28, CD30, CD69, GITR, CD95, CD178, CD152, IFN-γ, TNF-α, perforin and granzyme B. For functional studies, DNTs were purified from PBMCs through a negative selection and then cultured for 2 weeks. Data were acquired by 8-colour flow cytometer and analysed using Kaluza software. Other immune cells such as DCs, MDSCs and Treg was also detected to evaluate the correlation with DNTs. 7 cases of LNs received a finally diagnosis of reactive follicular hyperplasia (RFH) so they were considered as controls. All pts provided their informed consent in accordance with the Declaration of Helsinki. We observed a significant decrease (p =0.006) of circulating αβ-DNTs in untreated Ly pts (20.5 cells/ul ± 4.8) as compared with healthy controls (31.3 cells/ul ± 3.4) (fig.1-2) and their number seemed to be modulated during the follow-up. Their frequency in 1-month post-cht or disease relapsed pts was significantly decreased (p=0.006) as compared with the diagnosis as well as when compared indolent with aggressive histotypes (p=0.0001). In HL pts the frequency of αβ-DNT was significantly increased as compared with other histotypes (p=0.005) (Fig.3) Furthermore, the αβ-DNTs in LNs of Ly pts were significantly reduced as compared to to RFH-LNs (p=0.006) (fig.5) and are related to the aggressiveness of the disease. When evaluated either in MM and RRC pts the αβ-DNTs were significantly decreased (p=0.001) as compared with healthy controls and more interestingly when compared with Ly pts (p=0.001) given the greater immunological impairment of RCC/MM tumor burden (fig.6). Finally, ex vivo expanded DNTs acquired an immunomodulatory cytokine profile, characterized by the secretion of IFN-gamma and granzyme B, which are known as central components of anti-tumor immune responses supporting the hypothesis of αβ-DNT potential use in immunotherapeutic strategies (fig.4) Our preliminary data, showed an inverse correlation between the frequency of circulating αβ-DNTs and the tumor condition as well as that they could play an important role in both the development and the progression of lymphomas. This is the first study that compares the frequency of αβ-DNTs in three different pathologies correlating to tumor burden. In addition, it is likely that ex-vivo expanded DNTs exert an anti-tumor activity thus suggesting their possible use as a new strategy for adoptive immune-therapy. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Joh, Jae Won, Angel Callejas, Wendy Wong, Harvey Joel Cohen, Kari Nadeau, and Michael Jeng. "Role of Fas/FasL Pathway in Pediatric Idiopathic Neutropenia." Blood 110, no. 11 (November 16, 2007): 3291. http://dx.doi.org/10.1182/blood.v110.11.3291.3291.

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Abstract Idiopathic neutropenia in children is marked by low neutrophil counts (<1500/ul) circulating in the peripheral blood in patients whose disease spontaneously develops unrelated to drugs, cancers, specific antibodies, or known genetic deficiencies. Neutrophils, PBMCs and plasma were purified/obtained from 24 subjects (n=17 acute and n=7 chronic) with idiopathic neutropenia, aged 3 months to 11 yrs. After screening a number of cytokines (IL-2, IL-4 IL-6, IL-8, IL-10, IFN-γ, TNF-α), growth factors (GCSF), cell death factors (Fas, FasL, Granzymes/Perforin), and chemokines (CCL5, XCL1, XCR1), QT-PCR studies of neutropenic subjects’ neutrophils indicated an up-to-14-fold increase in Fas transcripts compared to age-matched healthy control neutrophils. FACS analysis on patient neutrophils demonstrated increased expression of Fas (93%+/− 15%) compared to healthy control neutrophils (41%+/−11%). No increase in Fas surface expression was seen on PBMCs or on CD4+T cells from neutropenia patients compared to healthy controls. Studies of patient plasma showed increased FasL in acute and chronic patients (up to 40-fold higher). Increased IL-6 and IL-10 levels in plasma were another distinctive characteristic of neutropenic patients. Healthy control neutrophils incubated in neutropenic patient plasma for 4 hrs showed greater rates of apoptosis (evaluated by PI/Annexin; up to 38-fold higher) compared to incubation with healthy control plasma. Heat denaturation, IgG exclusion, and size separation studies suggest that the killing factor(s) is a heat-sensitive protein which is not IgG and has a MW between 35 and 100 kD. Blocking with anti-FasL antibodies incubated with patient plasma caused a statistically significant 5-fold to 11-fold decrease in neutrophil apoptosis, approaching the rates of healthy controls, implicating FasL as a major mediator of neutrophil regulation. Thus, the Fas/FasL pathway may play an important role in idiopathic neutropenia. Patient number Chronicity Age FasL (pg/ml) Anti-neutrophil Ab Fold increase in neutrophil death over control plasma Fold increase in apoptosis over control plasma 1 chronic 2 yrs 7±1 positive 6.5±0.7 8.5±0.8 2 chronic 18 mo. 6±1 positive 7.3±0.5 8.1±0.2 3 chronic 23 mo. 5±0.5 wk positive/neg 7.8±0.6 8.5±0.4 4 chronic 1 yr 5±2 positive 6.5±0.5 7.3±0.6 5 chronic 5 yrs 7±1 wk positive 6.1±0.9 2.7±0.2 6 chronic 20 mo. 10±1 negative 11.3±1.4 2.1±0.5 7 chronic 11 mo. 6±1 negative 6.1±0.8 5.7±0.6 8 acute 2 yrs 16±2 N/A 18.7±1.3 22.4±2.9 9 acute 2 yrs 18±3 negative 12.0±2.1 19.1±2.5 10 acute 5 yrs 4±1 negative 2.5±0.5 2.6±0.4 11 acute 11 mo. 3±0.6 N/A 1.5±0.3 2.1±0.2 12 acute 3 yrs 17±1 negative 15.3±1.2 19.2±0.8 13 acute 2 yrs 40±3 negative 22.9±1.4 38.4±2.4 14 acute 2 yrs 21±2 N/A 14.3±1.0 20.5±1.2 15 acute 8 mo. 10±0.9 N/A 9.6±0.8 12.0±1.6 16 acute 17 mo. 19±3 negative 21.3±2.2 26.7±3.5 17 acute 6 yrs 8±1 N/A 6.7±1.3 11.5±2.1 18 acute 3 yrs 9±2 N/A 7.2±2.6 8.8±1.5 19 acute 11 yrs 12±2 negative 14.8±0.9 17.4±1.9 20 acute 5 yrs 6±1 negative 3.4±0.6 5.2±0.7 21 acute 4 mo. 8±2 N/A 6.2±1.0 8.1±2.2 22 acute 3 mo. 27±3 N/A 21.4±3.5 39.4±2.4 23 acute 11 yrs 22±1 negative 17.2±3.0 24.3±2.8 24 acute 2 yrs 18±2 positive 15.8±1.2 25.0±1.7 Representative control N/A 4 yrs 1.3±0.4 N/A 1.2±0.3 0.8±0.4

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Htut, Myo, Ghislaine Gallez-Hawkins, Joycelynne Palmer, Xueli Liu, R. Spielberger, Pablo Parker, Len Farol, et al. "Costimulatory Molecule Profiles After Autologous Hematopoietic Cell Transplantation (HCT) in Multiple Myeloma (MM) Patients." Blood 118, no. 21 (November 18, 2011): 5108. http://dx.doi.org/10.1182/blood.v118.21.5108.5108.

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Abstract Abstract 5108 Introduction: Co-stimulatory signal is antigen non-specific and is provided by the interaction of costimulatory molecules expressed on the membranes of antigen presenting cells (APC) and T/NK cells. It plays as a complementary second signal to interaction of MHC molecules on APC and T cell receptors on T cells (first signal). Costimulatory molecules can be either inhibitory or stimulatory and may play a role in MM cell growth. Blockade of these pathways may provide a new therapeutic avenue. Background: Prior studies have shown up-regulation of inhibitory pathways such as CTLA-4 and down regulation of activating CD28 in MM patients, compared to healthy controls but there was no comprehensive characterization of costimulatory functions in MM patients. This is a pilot study assessing NK cell reconstitution and expression of inhibitory molecules (PD-1 and CTLA-4) and stimulatory molecules (ICOS, 4-1BB, CD28 and OX40) before and after autologous transplantation in MM patients. Methods: Twelve adult patients with MM undergoing auto-transplant who are able to provide written informed consent are included in the study. Median age is 56.5 years (range 36 – 67). Peripheral blood samples were taken on 3–21 days prior to autologous HCT, 14, 30, 60, 90, 180 and 360 days after HCT. At the time of the current analysis, all patients were followed up to 90 days after autotransplant conditioned with high dose melphalan. Median length of follow up is 90 days post autologous HCT. A Flowcytometry method was developed in 2 six color panels to identify NK & T cells (using antibodies to CD3, CD56 & CD16) and subsets of NK cells expressing different costimulatory molecules using specific antibodies to CD28, CD152(CTLA-4), CD278(ICOS), CD134 (OX-40), CD137 (4-1BB) and CD279 (PD-1). Each panel contained 200 ul of whole blood treated with RBC lysing buffer before staining with antibodies. Clinical data were collected for disease status before and after HCT as well as the presence of documented infections. One way ANOVA test and Kruskal-Wallis test (non-parametric) were used to analyze the data. Disease status for MM was defined as per International myeloma working group guidelines. Results: Very Good Partial Response or better (≥ VGPR) was seen in 5 patients before HCT and 8 patients after HCT (best response was taken if there are different disease status in a given patient after HCT). Partial Response or worse (≤ PR) was seen in 7 patients before HCT and 4 patients after HCT. One patient had a progressive disease. NK cell number was highest (20% of total lymphocytes) at d14 (p =0.011) compared to pre HCT, d60 and d90 levels and back to pre HCT level at d90 (Figure 1). No statistically significant difference in individual costimulatory molecule expression was noted between pre-HCT vs. post-HCT periods. NK cell numbers were higher in patients with ≥VGPR compared to patients with ≤PR at d30 (p =0.004) and d60 (p =0.028) (Figure. 2). There was no significant difference in individual costimulatory molecule expression between ≥ VGPR vs. ≤ PR groups. Higher number of NK cells were noted in patients with infections up to d90 (p=0.0081). In contrast, no significant difference in individual costimulatory molecule expression was seen between patients with infections vs. no infections. Conclusion: At present analysis, we can determine that NK cell recovery is shorter (60–90 days) as opposed to CD 4 + T cells which have been shown to take about 12 months to recover after autologous HCT. Higher NK cell number were seen in patients with better disease response. There were no differences in costimulatory molecule profiles pre and post HCT based on early disease response. However, we plan long term follow up to see if this is a predictor of relapse. Disclosures: Off Label Use: in administration of plerixafor. Krishnan:Celgene: Speakers Bureau; Millenium: Speakers Bureau.

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Broome, Catherine, James K. McCloskey, and Raffaele Girlanda. "Successful Management Of Calcineurin Induced Thrombotic Microangiopathy(TMA) With Eculizumab After Non- Renal Solid Organ Transplantation." Blood 122, no. 21 (November 15, 2013): 1078. http://dx.doi.org/10.1182/blood.v122.21.1078.1078.

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Abstract Thrombotic microangiopathy (TMA) is seen in up to 30% of patients receiving solid organ transplantation and almost always occurs in the setting of calcineurin inhibitor (CNI) therapy. The underlying pathophysiology of calcineurin induced TMA is poorly understood. Long term follow up in non renal transplant patients with TMA suggests that in spite of plasma exchange therapy the 1 year mortality following TMA is up to 70%. Between November 2010 and August 2012, 7 patients at our institution who underwent organ transplants ( 5 small bowel, 2 orthotopic liver) developed clinical and laboratory evidence of TMA while receiving CNI therapy. TMA was diagnosed from 3 to 13(median 11) months post transplant and none of the patients responded symptomatically or by laboratory parameters to a reduction in dose of CNI. Other unsuccessful therapies included substitution of other immunosuppressive agents (N=1) and 11 daily plasma exchanges (N=1). At the time of TMA diagnosis notable laboratory values included platelets 22-73 (median 46) K/UL, hemoglobin 4.5 to 8.1(median 6.9) GM/DL, serum creatinine 1.16-5.4 (median 2.66)MG/DL, LDH 262-2903(median 435) Units/L, and ADAMSTS13 37-137%. All patients had a negative DAT, schistocytes on peripheral smear and all but one had undetectable haptoglobin. ( Table 1 ) Clinical symptoms at diagnosis included nausea, vomiting, abdominal pain, fever, hypertension, cerebral vascular accident (N=1), acute coronary syndrome (N=1). None of the patients had evidence of graft rejection on biopsy of the transplanted organ at the time of TMA diagnosis however 2 patients with small bowel transplants had pathologic evidence of ischemic changes and vascular thrombi on biopsy of the small bowel graft.Table 1Comparison of Medians of TMA Laboratory ParametersMedian ValuesPreTransplantTMA Diagnosis4 weeks post eculizumabPlatelet count K/UL12046202Hemoglobin GM/DL11.06.99.0Serum Creatinine MG/DL0.832.661.7LDH Units/L174435322HaptoglobinNT<3190 Eculizumab is a monoclonal antibody which binds with high affinity to C5 and is highly effective in disorders associated with abnormalities in the regulation of complement such as paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome (aHUS) another TMA disease. Due to the clinical and laboratory similarities of post transplant CNI associated TMA and aHUS all patients in our series were treated with the standard induction dose of eculizumab 1200mg weekly for 4 weeks and 900mg every 2 weeks thereafter. Treatment duration ranges from 4 weeks to 107 weeks. All patients were successfully maintained on adequate immunosuppression with calcineurin inhibitor (tacrolimus in all cases) to inhibit graft rejection. All patients demonstrated a rapid and complete resolution of laboratory and clinical manifestations of TMA. After the fourth dose of eculizumab platelet counts ranged from 104-291(median 202), hemoglobin 8.1-10.9(median 9.0), serum creatinine 0.70-4.08(median 1.7), LDH 157-475(median 322) and haptoglobin 23-204(median 90). Only 1 of the 4 patients requiring dialysis at TMA diagnosis remained on dialysis at 4 weeks of therapy.( Table 1) No patients show evidence of recurrent TMA or increase in infectious complications on continued eculizumab plus calcineurin inhibitor therapy at greater than 2 years of eculizumab therapy. The excellent clinical and laboratory response of our patients to eculizumab strongly suggests a central role for complement dysregulation in the pathophysiology of calcineurin induced TMA. There are multiple theories regarding the mechanism by which CNIs induce complement dysregulation including: (1) an underlying genetic predisposition to complement dysregulation worsened or exacerbated by CNI therapy, (2) CNI therapy may induce widespread and significant endothelial damage which serves as a stimulus for chronic complement activation, or (3) chronic over stimulation of complement production secondary to CNI inhibition of T-cell function .While the mechanism remains to be elucidated the clinical implications seem clear: CNI induced TMA is mediated by complement and is treated very effectively with eculizumab allowing patients to continue on graft function preserving CNI therapy. Disclosures: Broome: Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Off Label Use: Eculizumab for the treatment of calcineurin induced TMA.

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Fleming, Patrick, Maggie Cheung, and David Sokol. "Complement-Mediated Thrombotic Microangiopathy: A Murky Presentation of a Rare Disease Entity." Blood 132, Supplement 1 (November 29, 2018): 5005. http://dx.doi.org/10.1182/blood-2018-99-119893.

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Abstract Complement-mediated thrombotic microangiopathy (TMA), also known as atypical hemolytic uremic syndrome (aHUS) is a rare, hereditary, progressive, life-threatening disorder caused by a disruption in regulation of the alternative pathway of the complement system. Eculizumab, a terminal complement inhibitor, has emerged as a first-line therapy, however data are limited to small case series (Brocklebank et al., 2017). Here, we present a diagnostically challenging case of complement-mediated TMA, who received eculizumab therapy with excellent hematologic response. A 68-year-old female with history of possible Sjogren's syndrome, migraine disorder, chronic fatigue syndrome, inflammatory colitis, hypertension, and poor medical follow up presented with 6-day history of severe fatigue, hematochezia, decreased urine output, dyspnea with exertion and anginal chest pain. 2 weeks prior, patient endorsed "flu-like" illness and had diffuse myalgias without fevers. Further history revealed ibuprofen usage of 800-1200 mg/day for several years. Shortly after admission, patient became severely agitated and confused with an attempt to elope from hospital. During diagnostic workup, labs were significant for hemoglobin 5.6 g/dL, platelets 57,000/uL, serum creatinine 6.6 mg/dL, BUN 101 mg/dL. Peripheral smear showed schistocytes and tear drop cells, low platelets without clumping, and hypochromic normocytic red cells. LDH of 2152 U/L, haptoglobin <34 mg/dL, and GI PCR was panel negative for E. coli O157 and Shiga-like toxin producing E. coli. She was presumed to have thrombotic thrombocytopenic purpura (TTP) as she presented with 4 of the 5 characteristic pentad, including microangiopathic hemolytic anemia, acute renal failure, thrombocytopenia, and severe neurologic findings. Patient received several PRBC transfusions, five plasma exchange treatments, hemodialysis and corticosteroids. She had initial improvement in platelet count and decrease in LDH with plasma exchange, however plateaued by day 5. Further testing revealed low complement C3 level of 51 mg/dL, low complement C4 level of 22.7 mg/dL, and pre-PLEX ADAMSTS13 level of 93%, suggesting complement-mediated TMA as the correct diagnosis. Patient was subsequently transferred to tertiary care center for initiation of eculizumab. Genetic testing was completed, notable for decreased Factor H and a heterozygous missense mutation in complement factor H of uncertain significance, only having been previously reported in a single patient with aHUS (Fremeaux-Bacci et al., 2013). She achieved excellent hematologic response with eculizumab evidenced by improved platelet count, haptoglobin, decreased LDH, however she unfortunately remained dialysis-dependent. Thrombotic microangiopathy (TMA) syndromes are overlapping entities which can be categorized by primary vs secondary etiology. Primary syndromes include TTP (hereditary or acquired), Shiga-toxin mediated HUS, drug-induced TMA and complement mediated TMA. Secondary causes include pregnancy-associated (pre-eclampsia/HEELP syndrome), malignancy, systemic infection, severe hypertension, autoimmune disorders like SLE, and complications from organ transplantation. When evaluating a patient with suspected TMA, it is important to correctly categorize their disease to guide appropriate treatment. Complement-mediated TMA results from a hereditary deficiency of regulatory proteins that restrict activation of alternative complement pathway. These proteins include complement factor H (CFG) and its related proteins (CFHRs), membrane cofactor protein (MCP), CFI. Instead, it may result from an autoantibody inhibiting CFH of CFI. The consequence of this up-regulation is uncontrolled damage to vascular endothelium and renal cells, which manifests as a characteristic pentad. In our case, a CFH gene mutation was identified and history revealed a flu-like illness preceding her hospitalization. It is plausible that this illness may have served as a "second-hit" via complement-amplification. (Asif et al., 2017). Alternatively, in this patient with history of inflammatory colitis and reported Sjogren's syndrome, subclinical autoimmune disorder may also have served as a trigger. This case presentation serves as a reminder to not overlook the "zebra" that is complement-mediated TMA, to allow for prompt initiation of eculizumab therapy. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Xavier-Ferrucio, Juliana, Xiuqi Li, Vanessa Scanlon, Ping-Xia Zhang, Nadia Ayala-Lopez, Toma Tebaldi, Stephanie Halene, Karin E. Finberg, and Diane S. Krause. "Low Iron Promotes Megakaryocytic Commitment of Megakaryocytic-Erythroid Progenitors in Human and Mice." Blood 132, Supplement 1 (November 29, 2018): 2. http://dx.doi.org/10.1182/blood-2018-99-115124.

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Abstract The mechanism underlying thrombocythemia in patients with iron deficiency anemia remains unknown. Iron metabolism is highly conserved in humans and mice. We used Tmprss6-/- knockout (KO) mice as a genetic model of chronic iron deficiency anemia to study the effects of iron deficiency on lineage commitment. Tmprss6 is expressed in the liver, but not in hematopoietic cells, and is required for iron uptake from the diet. We observed that Tmprss6-/- mice have elevated platelet counts compared to littermate controls (average x1000/uL ± SD in wild type (WT): 477.9 ± 53.68, Tmprss6-/-: 915.9 ± 57; p<0.001; males and females 6-10 weeks old). Based on immunophenotype, the percentage of hematopoietic stem cells (HSC), megakaryocytic (Mk)-biased HSC (CD41+), megakaryocytic-erythroid progenitor (MEP), (Mk)- or erythroid (E)-committed progenitors in bone marrow (BM) is comparable between the two genotypes. To address whether thrombocythemia in iron deficiency anemia is related to altered commitment of MEP, we assayed the effects of low iron conditions on the function, transcriptome and signaling in MEP. CFU assays of single MEPs showed a striking increase in CFU-Mk (unilineage committed Mk progenitors) within the MEP gate in Tmprss6-/- at the expense of the bipotent CFU-Mk/E compared to WT (p<0.001). Thus, MEP in KO mice are significantly biased toward the Mk lineage, suggesting that low iron content may reprogram cell commitment. Consistent with this reprogramming effect, MEPs from KO mice have a 50% decrease in the labile iron pool (p=0.03, measured by Calcein-AM), and a 30% decrease (p=0.04) in proliferation relative to WT as measured by CFSE. To test the effect of altered iron content in vivo, we transplanted WT or KO BM into sublethally irradiated WT or Tmprss6-/- recipients. Tmprss6-/- recipients (of WT or KO BM) exhibited microcytic anemia and hypoferremia when compared to WT recipients (of WT and KO BM). Interestingly, the Mk-bias observed in non-transplanted Tmprss6-/- MEPs was recapitulated in Tmprss6-/-recipients (regardless of transplanted BM genotype) 6 months post-transplant (p < 0.009, n= 3 per group). We show using index single cell sorting followed by CFU assays that in WT mice, surface expression of transferrin receptor 1 (CD71) is a predictor of cell fate; cells from the MEP gate that grow as BFU-E have a higher CD71 mean fluorescence intensity (MFI) (179.9 ± 38.9) than cells that grow bipotent CFU-Mk/E colonies (65.9 ± 13.9) or Mk-only (CFU-Mk) colonies (24.9 ± 7.06; p<0.006). However, this correlation is lost in KO recipients, where the CD71 expression is elevated in all cells compared to WT (MFI: 279.9 ± 49.81, p<0.0001). RNAseq of WT and KO MEPs showed that 137 genes are differentially expressed (fold change: 1.5; p<0.05) between the two genotypes; of these, 84 are up regulated (e.g. Tfrc and Mybl2) and 53 are downregulated in the KO compared to WT. Surprisingly, mRNA encoding transcription factors that are known to regulate E vs. Mk maturation such as Myb, Tal, Fli1, and Eklf were unchanged. Pathway analysis of the differentially expressed genes highlighted metabolic pathways (asparagine and cholesterol biosynthesis and b-alanine degradation, p<0.008). We then asked if the iron regulation of the MEP commitment observed in iron deficient mice was also true for human MEP. In order to disrupt the iron sensing pathways in primary human MEP, we performed shRNA-based knockdown of Transferrin Receptor 2 (TFR2) using two different sequence targets and found a similar decrease in proliferation (p=0.03) and skewing toward Mk commitment (p<0.04) as was observed in Tmprss6-/- MEPs. Signal transduction analyses revealed that both human and murine MEPs in iron deficient conditions have lower levels of phospho-ERK (Thr202/Tyr204) compared to WT or scrambled controls. Regulation of erythroid differentiation by iron is thought to prevent inappropriate iron utilization when body stores are limited. To function in a protective manner, this checkpoint must act at stages prior to erythropoietin-mediated expansion of erythroid progenitors. Here we propose a model where iron acts on the bipotent progenitor, one possible source of Mk and E cells, to bias lineage commitment. These data support that low iron in the environment affects MEP metabolism, leading to lower ERK phosphorylation, slower proliferation and increased Mk commitment. Disclosures No relevant conflicts of interest to declare.

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Ezzat, Hatoon, Jonathan C. Wong, Douglas Filipenko, Linda M. Vickars, Paul F. Galbraith, Charles H. Li, Kevin Murphy, et al. "Improvement in Outcome of Patients with CD30+ HIV-Related Systemic B-Cell Non-Hodgkin’s Lymphoma (NHL) Providing HIV Infection Is Adequately Controlled." Blood 108, no. 11 (November 16, 2006): 4667. http://dx.doi.org/10.1182/blood.v108.11.4667.4667.

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Abstract Aberrant expression of the cell surface marker CD30 in systemic B-cell HIV-related NHL other than anaplastic large cell lymphoma (ALCL) has been reported. CD30 is a member of the tumor necrosis factor (TNF) family of receptors, and activation of CD30 mediates cell cycle regulation in preclinical models. The outcome of AIDS-related lymphoma (ARL) patients (pts) has improved dramatically in recent years with good control of HIV infection and the ability to intensify chemotherapy appropriate to the lymphoma. To determine whether CD30+ systemic ARL might have different clinical outcome and require a tailored approach to therapy, we performed a retrospective clinical-pathological correlation in pts seen at St. Paul’s Hospital in Vancouver, Canada (SPH) and focused on 39 B-cell lymphoma (BCL) pts since 1992. Clinical and HIV-related data were abstracted by chart review and from the CFE data-base. Stored biopsy material diagnostic of BCL was sectioned and stained for CD30 expression by immunohistochemistry. Median age was 41 (range 20–64) years and 37 pts were male. 16 pts had diffuse large B-cell lymphoma (DLBCL), 17 other B-cell NHL excluding ALCL, and 6 Hodgkin’s Lymphoma (HL); 29 were advanced stage. HIV risk was sexual in 31 and injection drug use (IDU) in 8. Median CD4 at diagnosis was 140 (10–770) cells/ul and 20 pts received HAART. Lymphoma therapy was administered according to era and histology: CHOP-like ± Rituximab (R) n=28; ABVD-like n= 6; CODOX-M/IVAC-R n=1; HAART alone n=1; resection n=1; declined therapy n= 2. 8 pts received added R and 4 added radiation therapy. 20 were CD30+ and 19 CD30−; the only baseline characteristic that differed significantly according to CD30 status was histology p<0.02 (6 of 6 HL were CD30+), no other HIV or lymphoma related characteristics differed by CD30 expression. In a Kaplan-Meier analysis, there was a trend toward improved overall survival (OS) in pts with CD30+ NHL; median survival (MS) was 16.3 (2.7–74.3) months (mo) for CD30+ vs. 5.9 (0–50) mo for CD30− (p<0.09). However, OS for HL was 100% at 11–66 mo vs. 7.2 (2.5–48) mo for DLBCL and 7.4 (0–74.3) mo for other NHL (p<0.04); when HL were removed from the analysis there was no difference in OS according to CD30 status (p<0.09), however progression-free survival (PFS) for all pts was; CD30+, 5.4 (0.8–20) mo and CD30−, 3.9 (0.03–6.2) mo (p<0.05). PFS for pts with a CD4 count at NHL Dx was; CD4<100, 3.3 (0–8.8) mo and CD4 ≤100, 5.9 (2.1–20) mo (p<0.04). PFS by CD4 count and CD30 status were as follows: CD4<100 and CD30+, 3.3 (0.8–8.8) mo; CD4<100 and CD30−, 2.0 (0.03–6.0) mo; CD4 ≤100 and CD30+, 6.7 (5.4–20) mo; CD4 ≤100 and CD30−, 4.5 (2.1–6.2) mo, (p<0.07). In conclusion, in this retrospective analysis, there was a trend toward improved PFS in pts with CD30+ B-cell NHL, particularly in pts with a CD4 count of at least 100. These data suggest that non-ALCL CD30+ NHL patients do not require intensified lymphoma therapy and underscores the importance of adequate control of HIV infection in the outcome of HIV-related lymphoma.

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Nearman, Zachary P., Marcin Wlodarski, Chris Hung, Evan Howe, Yadira Narvaez, Edward Ball, and Jaroslaw P. Maciejewski. "Immunogenetic Factors Determining Evolution of T-Cell Large Granular Lymphocyte Leukemia and Associated Cytopenias." Blood 106, no. 11 (November 16, 2005): 2211. http://dx.doi.org/10.1182/blood.v106.11.2211.2211.

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Abstract T cell large granular lymphocyte leukemia (T-LGL) is a chronic clonal lymphoproliferation of cytotoxic T cells (CTL). T-LGL presents with cytopenias, implying a relationship to other immune-mediated bone marrow failure states. T-LGL is often accompanied by autoimmune diseases, suggesting a clonal transformation in the context of an initially polyclonal autoimmune response. Immunogenetic predisposition factors have been described for both immune-mediated bone marrow failure as well as other autoimmune conditions and it is possible that some of these factors can promote evolution of T-LGL and/or the development of cytopenias. Here, we analyzed the association of T-LGL (and clinical variants) with a number of immunogenetic factors, including HLA and KIR genotype, KIR-L/KIR mismatch, CTLA4 (−658T/C, −318C/T, +49A/G) and cytokine single nucleotide polymorphisms (SNP) including: TNF-a (−308G/A), TGF-b 1 (codons 10 C/T, 25 G/C), IL-10 (−1082 G/A, −819 T/C, −592 C/A), IL-6 (−174 C/G), and IFN-g (+874 T/A). We studied a large cohort of patients with T-LGL (N=79) systematically followed at our institution. Using molecular analysis of TCR utilization as previously described (Wlodarski et al, Blood 2005) we have determined the relative and absolute size of the clonal LGL population for each case. We performed HLA typing as certain alleles serve as KIR ligands and can determine quality of immune response. HLA-B37 and Cw-7 were over expressed in LGL patients (13 vs 2%, p<.001; 60 vs 45%, p=.039); no other alleles correlated with the disease. No HLA association was found in patients presenting with neutropenia or splenomegaly, however HLA-Cw7 was found in 100% of patients with an increased number of clonal CTL (>1900/uL; N=13 vs 45% in controls and 33% in patients with low LGL count; p<.001). HLA-Cw alleles can be divided into C1 and C2 groups based on recognition by KIR; in the above 13 patients, C1/C1 phenotype was significantly increased compared to normal controls while C2/C2 was absent (p<.001, p=.04, respectively). This finding suggests a protective function for the C2/C2 phenotype in T-LGL patients. Subsequently, we analyzed KIR genotype by SSP PCR; no association between a specific KIR genotype and disease was found. LGL patients did not vary from controls in the ratio of inhibitory to stimulatory KIR. As KIR genotype may exert pathogenic influence in the context of KIR/KIR-L interactions, we hypothesized that a KIR/KIR-L mismatch would result in less regulation of CTL response and thus more severe cytopenias. According to the subdivision of HLA-Cw alleles into group C1 (ligands KIR2DL2 and 2DL3) and C2 (ligands 2DL1), genetic mismatch between KIR-L and KIR was found in 50% of patients, but proved statistically insignificant compared to normal controls. The severity of cytopenias and other hematologic manifestations was greater in patients without KIR-L/KIR mismatch. This finding may be due to an overshooting CTL response resulting from silencing NK-cells. SNP in cytokines and their promoters may play a pathogenic role in autoimmune conditions, as such we also analyzed the frequency of multiple cytokine polymorphisms in T-LGL to determine a potential secretion/stimulation phenotype. Among cytokine SNP studied in T-LGL, overrepresentation of TNF-a -308G/A polymorphism (for A/A: 14 vs 1% in controls, p=.009), and CTLA4 +49 SNP A/A genotype was found (50 vs 27%, p=.013), suggesting that the presence of G allele is protective.

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Sheth, Kruti, Raffaele Girlanda, and Catherine Broome. "Long-Term Management of Atypical Hemolytic Uremic Syndrome after Non-Renal Solid Organ Transplantation with Eculizumab." Blood 126, no. 23 (December 3, 2015): 3466. http://dx.doi.org/10.1182/blood.v126.23.3466.3466.

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Abstract Thrombotic microangiopathy (TMA) defined as microangiopathic hemolytic anemia, thrombocytopenia and end organ damage has been described in the post transplant (PT) setting after solid organ and hematopoietic stem cell transplants (HSCT). The true incidence among non-renal solid organ transplant (NRSOT) recipients is unknown. Reported rates range from 2-30% (Verbiest 2014). Despite standard interventions such as plasma exchange and/or reduction in calcineurin inhibitor (CNI) therapy, 3-month mortality rates remain as high as 40% in NRSOT patients who develop PT-TMA. In HSCT and renal transplant recipients, it has been demonstrated that complement plays a central role in the development of PT-TMA, analogous to atypical hemolytic uremic syndrome (aHUS) (Zuber 2011; Jodele 2013). Based on similar clinical manifestations and etiology, PT-TMA should be defined by the same laboratory criteria as aHUS and treated with eculizumab (ECU), a monoclonal antibody directed against C5 (Sheth, ASH 2014). We previously reported the successful use of ECU for the acute treatment of PT-aHUS in NRSOT patients (Broome, ASH 2013). We report the long-term data on this cohort of patients here. Between January 2012-Novemeber 2013, 10 patients (8 small bowel, 2 liver), were diagnosed with PT-aHUS utilizing established clinical criteria for de novo aHUS. All patients were on CNI therapy at the time of diagnosis. The median time from transplant to TMA diagnosis was 10.5 months (range 1-53 months). All patients received standard aHUS dosing of ECU (with meningococcal vaccinations and antibiotic prophylaxis). By 1 month of ECU therapy, all patients demonstrated normalization of hematologic parameters and 4 of the 5 patients on hemodialysis (HD) at the time of ECU initiation were able to discontinue HD. At a median follow-up of 24 months, 7 patients remain on therapy (range 18-43 months). One patient had ECU discontinued at 3 months by the transplant team. Two patients have died: one at 3 months of therapy due to sepsis/multi-organ failure and the other at 6 months of therapy due to PT lymphoproliferative disorder/graft failure (GF). The 7 patients remaining on ECU had no clinical/laboratory evidence of recurrence of PT-aHUS (Table 1). No new HD has been initiated in the follow-up period. All remained on CNI therapy (tacrolimus) for prevention of transplant rejection. One patient on ECU therapy developed GF. No serious infections have been reported to date. ECU is an effective therapy for the long-term management of PT-aHUS in NRSOT recipients. Prompt initiation of ECU appears to result in an increased likelihood of reversal of end organ damage related to TMA. The 2 patients with GF (240 days, 572 days) and the 1 patient who remained on HD (180 days) had extended intervals from laboratory evidence of PT-aHUS to initiation of ECU, suggesting prolonged complement mediated TMA may contribute to irreversible organ damage. With early ECU therapy, 86% of patients have healthy graft function and 80% of patients were able to discontinue HD. Despite ongoing CNI therapy, renal function, as measured by serum creatinine, has continued to improve demonstrating the benefit of long-term ECU therapy. The observed decline in haptoglobin, an acute phase reactant, at 12 and 18 months suggests a decrease in systemic inflammation with constant complement regulation mediated by ECU, illustrating the systemic nature of PT-aHUS. All patients remained on both CNI and ECU without infectious complications, demonstrating the ability to successfully administer more than one immune mediator in NRSOT patients with PT-aHUS concurrently. The mortality rate in our cohort of NRSOT recipients with PT-aHUS is 20%, significantly less than prior reports of up to 70%. We propose treatment with ECU at the earliest clinical/laboratory signs of aHUS to prevent the morbidity and mortality associated with PT-aHUS in NRSOT patients. Future studies should prospectively evaluate ECU therapy in PT-aHUS to better define the risks and benefits of this therapy in NRSOT patients. Table 1. Median Laboratory Values Post ECU At diagnosis 1 month 3 months 6 months 12 months 18 months Platelets k/uL 86 151 p=.01 175 p=.04 177 p=.01 191 p=.04 168 p=.07 Serum Creatinine mg/dL 2.26 1.54 p=.03 1.42 p=.05 1.28 p=.04 0.97 p=.13 1.18 p=.13 LDH units/L 369 302 p=.23 206 p=.17 186 p=.17 235 p=.23 158 p=.21 Haptoglobin mg/dL <8 115 p=.00 154 p=.01 136 p=.02 67 p=.03 90 p=.04 Disclosures Broome: Alexion Pharmaceuticals: Consultancy, Honoraria, Research Funding.

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Hod, Eldad A., Richard O. Francis, Steven L. Spitalnik, Aaron Winters, Keegan S. Cooke, and Barbra J. Sasu. "Validation and Preclinical Correlation of a New Sandwich ELISA for Measuring Murine Hepcidin." Blood 120, no. 21 (November 16, 2012): 2100. http://dx.doi.org/10.1182/blood.v120.21.2100.2100.

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Abstract Abstract 2100 Although enzyme-linked immunosorbent assays (ELISA) exist for measuring human hepcidin, no commercially available antibodies to mouse hepcidin are validated for this purpose. Thus, we developed an ELISA to measure mouse hepcidin using low plasma volumes to allow for repeat testing of individual mice over time. To this end, human hepcidin knock-in mice (Hep2 mice) were used to generate a panel of anti mouse hepcidin antibodies. A sandwich ELISA using these antibodies was validated. Pooled plasma from iron-deficient (combining diet and phlebotomy), iron-replete, and inflamed (injecting sub-clinical amounts of lipopolysaccharide) FVB mice was used to prepare frozen aliquots of low, medium, and high reagent control plasma. These samples were tested on 5 different days (n=8 wells/sample/day). Mean hepcidin levels for the high, medium, and low reagent were 791, 504, and 117 ng/mL, respectively. The intra-assay coefficient of variation (CV) was 10, 4.5, and 15.6% for the high, medium, and low samples, respectively. Inter-assay CV was 4.7, 4.0, and 11.1% for the high, medium, and low samples, respectively. Analytical sensitivity was 0.7 ng/mL and the assay was linear (Pearson r=0.99) to 500 ng/mL at a 1:100 dilution of sample to blocking buffer (i.e. only 1 uL of sample needed per test). With 20 plasma samples spanning the range of expected hepcidin levels, there was high correlation between hepcidin determined by ELISA and the sum of hepcidin-1 and hepcidin-2 by time-of-flight mass spectrometry (Pearson r = 0.99; P<0.0001). Stability was analyzed using samples from repeated freeze-thaw cycles at −80°C or from incubation at 4°C. Each freeze-thaw cycle induced up to a 15% loss in hepcidin. Overnight refrigeration led to a 31%, 21%, and 4% decrease in hepcidin for the high, medium, and low samples, respectively. Severe hemolysis (i.e. 8 g/dL hemoglobin) caused interference, decreasing apparent hepcidin levels up to 52%; mild hemolysis (0.1 g/dL hemoglobin) decreased hepcidin levels up to 15%. To provide clinical correlation in mouse models, we used cohorts (n=5/group) of C57BL/6 females with differing iron status: Iron deficient: weanling mice on a low iron diet (0–5 ppm FeSO4; AIN diet) for 8 weeksIron-replete: weanling mice on a normal iron diet (45 ppm FeSO4; AIN diet) for 8 weeksIron-overload: mice received weekly iron-dextran injections (5 mg for 7 weeks)Transfusional iron-overload: mice received weekly syngeneic RBC transfusions (1 unit for 7 weeks) Hepcidin mirrored ferritin levels and total liver iron measured at necropsy (see Table). To test whether this ELISA can track hepcidin during infection, and whether iron status affects this response, iron-deficient or -replete mice (n=10/group) were inoculated with 1000 colony forming units of Salmonella typhimurium and blood samples obtained pre-infection and 1–7 days post-infection to measure hepcidin and interleukin (IL)-6. Pre-infection, iron-deficient mice had significantly lower hepcidin levels than iron-replete mice (<0.7 vs. 136 ± 56 ng/mL, mean ± sd). On Day 7 post-infection, IL-6 was much higher in surviving iron-deficient mice (up to >2000 pg/mL) than in iron-replete mice (207 ± 70 pg/mL). Despite dramatic increases in IL-6, hepcidin increased less in iron-deficient mice (260 ± 132 ng/mL) than in iron-replete mice (636 ± 273 ng/mL). Thus, despite a stronger inflammatory stimulus, hepcidin levels are muted in iron-deficient animals. In conclusion, this validated ELISA is a new tool that can be used to study iron regulation in mice. Liver iron (μg) Ferritin (ng/mL) Hepcidin (ng/mL) Iron replete 62 ± 10 849 ± 100 204 ± 32 Iron deficient 31 ± 5 387 ± 114 2.4 ± 1 Iron overload (Fe-Dextran) 11531 ± 1163 109912 ± 11872 633 ± 54 Iron overload (Transfusion) 352 ± 1 3302 ± 872 323 ± 64 Data presented as mean ± sd. Disclosures: Winters: Amgen, Inc.: Employment. Cooke:Amgen, Inc.: Employment. Sasu:Amgen, Inc.: Employment.

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Feng, Di, Huanping Zhou, Xiaohong Jin, Juan Wei, Qingqing Zhang, Yang Gu, Pengcheng Zhang, et al. "Electroacupuncture Pretreatment Alleviates LPS-Induced Acute Respiratory Distress Syndrome via Regulating the PPAR Gamma/NF-Kappa B Signaling Pathway." Evidence-Based Complementary and Alternative Medicine 2020 (July 23, 2020): 1–10. http://dx.doi.org/10.1155/2020/4594631.

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Electroacupuncture (EA) is reported to possess anti-inflammatory properties and has beneficial effects on acute respiratory distress syndrome (ARDS). However, the underlying mechanisms of the effects of EA on ARDS remain unclear. This study aims to investigate the protective effect of EA on LPS-induced ARDS. In this study, Sprague-Dawley male rats were treated with EA at Hegu (LI4) for 45 minutes before LPS instillation (0.4 mg/kg, 100 ul). H&E staining, wet-to-dry weight (W/D) ratio, PaO2, and protein content in BALF were employed to determine the function of lung tissues. Inflammatory cytokines in serum and BALF were detected by enzyme-linked immunoassay assay (ELISA). The levels of oxidative stress markers were detected to determine the oxidative stress status. Cell apoptosis was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and western blot. Here, we found that EA pretreatment effectively alleviated lung pathological damage. Moreover, EA suppressed the oxidative stress damage by upregulating glutathione and superoxide dismutase and downregulating malondialdehyde. EA pretreatment also regulated apoptosis-related proteins, such as Bax and Bcl-2. We found that peroxisome proliferators-activated receptors γ (PPARγ) play a critical role during ARDS, EA up-regulated the expression of PPARγ, which inhibited the activation of nuclear factor-kappa B (NF-κB) and decreased the inflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α). When rats were treated with GW9662, a selective PPARγ antagonist, these effects of EA were reversed. Our study demonstrated that EA pretreatment had a beneficial effect on LPS-induced ARDS in rats by anti-inflammatory, antioxidative, and antiapoptotic properties which was regulated via PPARγ/NF-κB signaling pathway.

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Zhou, Zhou, Hua Jing, Zhenyin Tao, Huiwan Choi, Khatira Aboulfatova, Renhao Li, and Jing-fei Dong. "Effects of Naturally Occurring Mutations in CUB-1 Domain on ADAMTS-13 Synthesis, Stability and Activity." Blood 110, no. 11 (November 16, 2007): 710. http://dx.doi.org/10.1182/blood.v110.11.710.710.

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Abstract Upon stimulation, vascular endothelial cells release von Willebrand factor (VWF) in the unusually large (UL) and prothrombotic forms. The hyperactive ULVWF multimers are rapidly cleaved by the metalloprotease ADAMTS-13 to smaller forms that are hemostatically active, but no longer prothrombotic. Lack of this VWF- cleaving activity results in accumulation of ULVWF, which agglutinates platelets, leading to microvascular thrombosis as demonstrated in patients with thrombotic thrombocytopenia purpura (TTP). ADAMTS-13 deficiency seen in TTP results from either mutations in the ADAMTS13 gene in familial cases or autoantibodies against the metalloprotease in acquired cases. ADAMTS-13 differs from other members of the family metalloproteases by containing two C-terminal CUB domains, which contain the binding site for VWF and whose mutations are associated with TTP. However, recombinant ADAMTS-13 without CUB domains remains active in vitro. To study the functional role of CUB domains, three naturally occurring mutations in the CUB-1 domain (C1213Y, W1245del and K1256frameshift, the latter two removed CUB-2 and the C-terminal part of CUB-1 domain) were generated by site-directed mutagenesis and expressed in the mammalian Hela cells. The release, stability, and activity of the mutants were then examined. We found that the mutations significantly reduced the release of recombinant ADMATS-13 into the culture medium, but not to the extracellular matrix. The intracellular pools of the recombinants detected in cell lysates were comparable to that of wild-type, indicating that the mutations affected the targeted secretion of the metalloprotease. When measured during a course of up to one month at −80°C, C1213Y and K1256framshift significantly accelerated, whereas W1245del delayed, degradation of the recombinants, which was inhibited by 5 mM of EDTA. The observation demonstrates that CUB domains are also critical for the structural stability of the metalloprotease. In comparison, the three mutants remained active in cleaving (UL)VWF under static and flow conditions. Consistent with the activity measurements, all three mutants bound immobilized VWF with similar kinetics in ELISA assay, indicating that the N-terminal sequence (C1192-D1212) of CUB-1, which is not affected by the mutations, contains the binding site for VWF. The conclusion is supported by our previous data that the CUB-1 synthetic peptides derived from the N-terminal region (H1196-P1220) blocked VWF-ADAMTS-13 interaction and cleavage of ULVWF under flow. Together, these results demonstrate that the CUB domains (primarily CUB-2 and the C-terminal part of CUB-1) are crucial for regulating the targeted ADAMTS-13 secretion and the stability of the metalloprotease; the N-terminal sequence of CUB-1 domain contains the VWF binding site that remains intact after deleting the rest of CUB domains; and the primary cause of ADAMTS-13 deficiency found in patients carrying these mutations is likely due to defective secretion of the metalloprotease.

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Hopkins, Courtney K., Christian Riley, Samuel Pepkowitz, and Jean R. Lopategui. "Absence of the V617F JAK2 Mutation in 181 Healthy Donors." Blood 110, no. 11 (November 16, 2007): 4664. http://dx.doi.org/10.1182/blood.v110.11.4664.4664.

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Abstract INTRODUCTION: Janus kinase 2 gene (JAK2) encodes for a cytoplasmic tyrosine kinase involved in normal hematopoietic growth factor signaling. Point mutations of the JAK2 gene on chromosome 9, specifically V617F, a point mutation at amino acid 617, are associated with myeloproliferative disorders (MPD). The V617F JAK2 mutation has been found in 90% of patients with polycythemia vera, 50–60% of patients with essential thrombocythemia or idiopathic myelofibrosis and 1–5% of patients with other MPD. To our knowledge, previous studies involving the V617F JAK2 mutation were not performed on a control population of normal individuals. Therefore, the prevalence of this mutation has not been established. In this study, we tested volunteer blood donors from a hospital-based blood donation center for the presence of the V617F JAK2 mutation. METHODS: Citrated whole blood was obtained from volunteer blood donors, age 17 and older, who presented to donate whole blood at a hospital-based blood donation center. The donors met all qualifications to donate blood as defined by FDA regulations. DNA was extracted using the QIAagen and QIAamp DNA extraction columns, quantified and diluted to 100ng/ul. DNA was simultaneously amplified and detected using allele specific minor groove binder probes and primers for the V617F JAK2 mutation. The resultant amplification was recorded by real-time, quantitative PCR using an ABI 7500 (Applied Biosystems, Foster City, CA). A 1% limit of detection, determined from sensitivity and specificity studies using a known cell line control, was set as the technically reproducible threshold sensitivity of the test. Samples were defined as negative for the V617F JAK2 mutation if only the wild type allele was detected. Samples that had a mutant allele detected above the 1% limit of detection were defined as positive for the V617F JAK2 mutation. Samples that had a mutant allele detected below the 1% limit of detection were defined as negative for the V617F JAK2 mutation. RESULTS: A total of 181 DNA samples from volunteer blood donors were tested for the V617F JAK2 mutation. The test group consisted of 104 males (mean age 44, range 17–77) and 77 females (mean age 42, range 18–71). Of the 181 donors tested, 171 had only wild type allele detected and were considered negative. Ten donors had high background of the mutant allele detected below the 1% limit of detection and were considered negative. DISCUSSION: To our knowledge, this is the first report documenting the prevalence of the V617F JAK2 mutation in a healthy blood donor population. In this study of 181 volunteer blood donors none had the V617F JAK2 mutation. Although 10 of the 181 donors were found to have mutant allele detected, they were below the 1% technically reproducible sensitivity threshold of the test and were considered negative. We recommend that mutations detected below the technical threshold of 1% of our assay be considered false positives. The results of this study suggest that the V617F JAK2 mutation is not present in a healthy blood donor population and is significant when detected by our method.

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Hopkins, Courtney K., Christian Riley, Samuel Pepkowitz, and Jean Lopategui. "Absence of FLT3 Mutations in 181 Healthy Blood Donors." Blood 110, no. 11 (November 16, 2007): 4273. http://dx.doi.org/10.1182/blood.v110.11.4273.4273.

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Abstract INTRODUCTION: FMS-like tyrosine kinase 3 gene (FLT3) encodes for a tyrosine kinase receptor located on hematopoietic progenitor cells in the bone marrow, lymph nodes and thymus. Activation of FLT3 results in increased proliferation of these hematopoietic cells. Mutations in the FLT3 gene, resulting in a constitutively activated FLT3 receptor, have been identified in 1/3 of patients with acute myelogenous leukemia and portend a worse prognosis. The most frequently occurring FLT3 mutations are internal tandem duplications (ITD) located in the juxtamembrane domain. To our knowledge, previous studies involving FLT3 mutations have not been performed on a control population of normal individuals. Therefore, the prevalence of this mutation has not been established. In this study, we tested volunteer blood donors from a hospital-based blood donation center for the presence of FLT3-ITD mutations. METHODS: Citrated whole blood was obtained from volunteer blood donors, age 17 and older, who presented to donate whole blood at a hospital-based blood donation center. The donors met all qualifications for donating blood as defined by FDA regulations. DNA was extracted using the QIAagen and QIAamp DNA extraction columns, quantified and diluted to 100ng/ul. DNA was amplified using two fluorescently labeled primers for the FLT3-ITD mutation (InVivo Scribe Technologies, San Diego, CA). Using an experimental assay with high sensitivity but high non-specific background, FLT3-ITD mutations were detected by means of size and color separation through capillary electrophoresis (Vidiera NsD, Beckman-Coulter, Fullerton, CA). Samples were defined as negative for a FLT3-ITD mutation if only a single 330 base pair (bp) peak was detected. Samples were defined as positive for a FLT3-ITD mutation if an additional second peak > 330 bp was detected. Samples that had a small second peak (<5000 relative fluorescent units (RFU)) on initial testing were repeated using a highly specific protocol with low non-specific background. Samples that on repeat testing did not demonstrate the second peak were defined as negative for a FLT3-ITD mutation. RESULTS: A total of 181 DNA samples from volunteer blood donors were tested for FLT3-ITD mutations. The test group consisted of 104 males (mean age 44, range 17–77) and 77 females (mean age 42, range 18–71). Of 181 donors, 177 were negative for FLT3-ITD on initial testing. Only 4 donors (2 females: 18 and 19 years old and 2 males: 51 and 59 years old) had very small (<5000 RFU) second peaks > 330 bp. However, these second peaks were not detected with repeat testing and were therefore considered negative. DISCUSSION: To our knowledge, this is the first study documenting the prevalence of FLT3-ITD mutations in a healthy population. In this study of 181 volunteer blood donors none had FLT3-ITD mutations. Although 4 of the 181 donors were initially found to have very small additional second peaks equivocal for FLT3-ITD mutations, all were negative with high specificity, low background repeat testing, indicating these were probably false positive results. Therefore, we recommend that very small additional second peaks should be confirmed with repeat testing. The results of this study indicate that FLT3-ITD mutations are not present in a healthy blood donor population and are therefore significant when detected in leukemic patients.

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Chen, Bao-An, Jie Xiong, Long Ba, Feng Gao, Chong Gao, Jia-Hua Ding, Yun-Yu Sun, et al. "Identification of Transcription Factor AP-1 Bound to Untranslated 5′ Regulatory Region DNA of mdr1 Gene with Atomic Force Microscopy." Blood 108, no. 11 (November 16, 2006): 4195. http://dx.doi.org/10.1182/blood.v108.11.4195.4195.

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Abstract Multidrug resistance(MDR) phenotype of cancer cells is a major obstacle for cancer chemotherapy, this phenotype is main due to the overexprssion of the mdr1 gene. Transcription factor AP-1, which is one of important regulate proteins in the promoter region of mdr1 gene. To date, no direct data of AP-1-DNA regulating complexes on mdr1 gene. A novel method for identifying DNA-binding proteins from image analysis using atomic force microscopy(AFM) was developed. This study is to image and map the structure of Untranslated 5′ regulatory region DNA of mdr1gene, identificate and analysis of transcription factor AP-1 bound to Untranslated 5′ regulatory region DNA of mdr1 gene complex with atomic force microscopy, to find out the molecule mechanism of multidrug resistance. Human leukemia adriamycin resistant strain K562/A02 was used as a target cells, transcription factor AP-1 was used as a target protein. Cultivate and PCR technique was used to amplify K562/A02 cells with the 769bp 5′regulatory region DNA of mdr1 gene, which fragment from −755 to +14. The amplified DNA products were purified by the PCR product purification kit, AP-1 of hela nuclear extract were purified by Sephadex spin-column filled with a bed of Sephadex G-100. The target DNA fragments were incubated with the target protein AP-1 at a 1:2 molar ratio in binding buffer and then immobilized the the AP-1-DNA complexes on a freshly cleaved mica surface which treated by MgCl2. AFM was used to idificate image of the structure of target DNA and the AP-1-DNA complexes. We show here the applicability of AFM in the quantitative analysis of the molecular mechanisms of DNA-protein interaction: The optimum DNA concentration to yield well-absorbed DNA molecular on the mica surface was found to be 10ng/ul. the contour of target DNA AFM image :the length of the DNA fragment measured by AFM image was 260.13± 2.29nm, the width was 11.88 ± 0.92nm, the mean height was 1.2nm(mean±SD, N = 50). Sephadex spin-column (Ultrafree- MC 0.22) can be used to purificate DNA and protein-DNA complex, the contour data of AP-1 protein AFM image:: the width of proteinlarge was 30±3.2nm, protein small was 19±2.8nm; the height of proteinlarge was 3.8±1.4nm, proteinsmall was 3.2±1.8nm (mean±SD, N = 30). we got the contour data of pro- tein-DNA complexes: the width of protein large was 28±2.7nm, the height was 3.4± 0.94nm (mean±SD, N = 8), the width of proteinsmall was 18±1.7nm, the height was 3.1±2.2nm (mean±SD, N = 22), and deduced the possible AP-1 site on mdr1DNA located between bases −126 and −115bp, this result is in close agreement with the expected −121 and −115 bp values. the overall connection efficiency of protein was about 10%. The AFM method to visualize individual biomolecules allows us to investigate the conformation of protein-DNA complexes.

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McGlauflin, Rose, Pan Li, Prudovsky Igor, Calvin Vary, and Pradeep Sathyanarayana. "Utilizing Exosomes As Innovative Microrna Biomarkers and Targeted Drug-Delivery Vehicles in Acute Myeloid Leukemia." Blood 126, no. 23 (December 3, 2015): 3595. http://dx.doi.org/10.1182/blood.v126.23.3595.3595.

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Abstract Background: Exosomes are microvesicles that play important roles in intercellular communications in both normal and tumor cells via their cargoes, which include microRNA (miRNA) and proteins. miRNAs play critical regulatory roles in hematopoiesis, and their abnormal expression is correlated with several hematological malignancies, including acute myeloid leukemia (AML). Exosome-derived miRNAs and proteins are increasingly recognized for their prognostic biomarker potential. Exosomes are being evaluated for their potential as novel drug-delivery vehicles due to their endogenous nature and ability to carry small molecule drugs. Aims: We aim to characterize HSC-derived exosomes, investigate the biomarker potential of exosomes derived from AML patient samples (by examining their proteomic and miRNA profiles) and utilize exosomes as innovative drug delivery vehicles with the ability to eliminate leukemic stem cells in a targeted manner. Methods: Secreted exosomes from murine bone marrow HSCs were isolated from conditioned medium and visualized using confocal microscopy. We isolated exosomes and performed miRNA profiling, using qPCR, and LC-MS/MS proteomic analysis to characterize the constituents. We also isolated exosomes from the CD34+ cells of three AML patient samples and profiled 372 of the most abundantly expressed miRs in these cells compared to normal CD34+ cells. We analyzed the proteome of these exosomes as well. In order to assess the utility of exosomes as drug carriers, exosomes from bone marrow-derived OP9 stromal cells were transfected with Daunorubicin (1ug/ul). Normal CD34+ cells and patient-derived AML samples (from n=2 patients) were treated with varying doses of the drug-loaded exosomes. Drug-loaded exosomes uptake was tracked with a Texas Red siRNA. After 24 hours, cells were screened for apoptosis. To test the feasibility of targeted exosomes, OP9 cells were exposed to AML patient samples for 48 hours. The patient cells were then removed and, 24 hours later, "trained" stromal cell-derived exosomes were isolated from the media and transfected with Daunorubicin. These patient-specific, exosomes were plated with both the corresponding patient's CD34+ cells as well as normal CD34+ primary cells. After 24 hours, apoptosis was measured. Results: miRNA profiling of murine bone marrow showed miR-21a, miR-92a and miR-25 were most abundant in exosomes. Proteomic LC-MS/MS analysis revealed presence of exosome-associated novel proteins such as Syntenin-1. Syntenin-1, which is known to bind IL-5R and promote myelopoiesis, was present in significantly higher levels in HSCs compared to myeloid progenitors, implying a functional role for exosome derived Syntenin-1. miRNA profiling in AML samples revealed distinct signature profiles. Importantly, exosome derived miRs such as -1290, -375, -205 and -21-that are known prognostic markers in cancers such as prostate, ovarian and hepatocellular carcinoma-were significantly upregulated in all the three exosome-derived AML samples. The drug-loaded exosomes were successful in inducing significant apoptosis in two patient samples tested. These drug-loaded exosomes also induced cell death in CD34+ normal cells when compared to control exosomes. However, the patient-trained exosomes specifically eliminated 92% of CD34+ AML patient cells, while causing significantly less cell death (44%) of normal CD34+ primary cells exposed to drug-loaded, patient-trained exosomes. Summary/Conclusion: Taken together, our data predict important functional roles for exosome-derived Syntenin-1 in regulating lineage specific hematopoietic differentiations. Furthermore, for the first time, we have identified highly upregulated select exosome-derived miRs from AML patient samples whose prognostic value has been recently reported for other cancers, making these miRs promising candidates for AML biomarkers as well. Finally, stromal cell-derived, drug-loaded exosomes are not only able to induce apoptosis in AML patient samples, but they can effectively be trained by leukemic cells to favor uptake resulting in targeted elimination of leukemic over normal CD34+ cells. Disclosures No relevant conflicts of interest to declare.

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Feng, Yumei, Wencheng Li, Hua Peng, and Atsuhiro Ichihara. "Abstract 25: Prorenin Elevates Blood Pressure via the (Pro)renin Receptor: Who Needs Renin When You Have Prorenin in the Brain." Hypertension 60, suppl_1 (September 2012). http://dx.doi.org/10.1161/hyp.60.suppl_1.a25.

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Growing evidence supports that the brain renin-angiotensin (Ang) system (RAS) plays an important role in blood pressure (BP) regulation and hypertension. This concept has been perplexed by extremely low renin activity in the brain. We recently reported the presence of prorenin in the mouse brain at a level of 10-fold higher than that of renin. In addition, we and others have identified the localization of the (pro)renin receptor (PRR) in various brain nuclei involved in BP regulation. The binding of prorenin to PRR leads to Ang II formation in vitro. Our hypothesis: Brain prorenin binding to PRR mediates Ang II formation, leading to an increase in BP. To test this, neuron-specific PRR knockout mice (Nefh-PRR) and wildtype (WT) littermates (N=5/group) were each implanted with a telemetric probe for BP recording and an intracerebroventricular (ICV) cannula for infusion of mouse prorenin (100ng/ul), mouse renin (100ng/ul), or Ang II type 1 receptor (AT1R) blocker (losartan, 10ug/ul) at 0.3ul/minute for 10 minutes. Mouse prorenin infusion increased the BP (mmHg) in WT mice (ΔMAP: 41 ± 5); however, the prorenin-induced pressor response was abolished in Nefh-PRR mice (ΔMAP: 5 ± 1). Infusion of mouse renin similarly increased BP in Nefh-PRR (ΔMAP: 27 ± 2) and WT (ΔMAP: 31 ± 5) mice. The pressor response induced by prorenin or renin was completely blocked by the infusion of losartan. The data suggests that ICV prorenin via PRR mediates Ang II-dependent pressor response in WT mice. To determine whether PRR contributes to the development of brain RAS-dependent hypertension, Nefh-PRR and WT littermates (N=8/group) were treated with 50mg of deoxycorticosterone acetate (DOCA) subcutaneously, plus 0.9% NaCl drinking water for 21 days. The baseline BP was similar between Nefh-PRR (101 ± 2) and WT (101 ± 3) mice. BP was increased in WT mice (132 ± 6) by DOCA-salt treatment, while Nefh-PRR mice remained normotensive (108 ± 3). In summary, prorenin via PRR mediates AngII/AT1R-dependent pressor response in the brain. Neuron-specific PRR deletion attenuates the development of DOCA-salt hypertension likely due to the lack of Ang II/AT1R activation. We conclude that prorenin/PRR may be the key factors to initiate the brain RAS and play an essential role in neurogenic hypertension.

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Haugen, Margaretha, Kristin Holvik, and Per Ole Iversen. "Evaluation of Tolerable Upper Intake Levels for Vitamin D in Children and Adolescents." European Journal of Nutrition & Food Safety, February 26, 2019, 102–3. http://dx.doi.org/10.9734/ejnfs/2019/v9i230046.

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In 2012, the European Food Safety Authority (EFSA) suggested a tolerable upper intake level (UL) for vitamin D at 100 µg/day for adults based on the risk of hypercalcaemia. EFSA concluded that consumption of up to 50 µg/day does not lead to hypercalcaemia in children and adolescents (10-17 years). Furthermore, EFSA stated that there is no reason to assume that children and adolescents in the phase of rapid bone formation and growth have a lower tolerance for vitamin D compared to adults, and a UL of 100 µg/day for adolescents aged 11-17 years and 50 µg/day in children 1-10 years, taking the smaller body size into account, was proposed. The Norwegian Food Safety Authority (NFSA) is currently revising the national regulation of maximum limits in food supplements (not yet harmonised in the European Economic Area (EEA)), including maximum limits for vitamin D. NFSA has therefore requested the Norwegian Scientific Committee for Food Safety (VKM) to evaluate the assumption in the EFSA opinion that children and adolescents can tolerate the same amount of vitamin D as adults due to rapid bone formation and growth. In children and adolescents with lower weight than adults, this assumption actually implies that adolescents can tolerate more vitamin D per kg body weight than adults. VKM is therefore requested to evaluate if there is scientific evidence that a UL at 50 µg/day for children (1-10 years) and 100 µg/day for adolescents (11-17 years) is safe. The present statement is prepared by members of the Panel on Nutrition, Dietetic Products and Novel Food and Allergy in VKM. Three literature searches were performed to find new relevant studies investigating high intakes of vitamin D in children and adolescents and the role of vitamin D in bone formation and growth. No studies supporting a higher tolerance to vitamin D in children and adolescents due to rapid bone formation and growth were retrieved in the literature search. Moreover, there is apparently no firm association between bone formation and vitamin D levels in children during their growth period into adolescence and adulthood. No studies investigating high intakes of vitamin D in children 1-10 years were found. Furthermore, no studies that have examined safety issues and/or adverse effects of vitamin D supplementation in doses above 50 µg/day in adolescents were identified. It can therefore not be concluded that the UL at 50 µg/day in children (1-10 years) and 100 µg/day in adolescents (11-17 years) is safe. In the 2002 report from European Scientific Committee on Food (SCF), a UL was set at 25 µg/day for children aged 2-10 years, and 50 µg/day for adolescents aged 11-17 years (corresponding to the UL for adults at that time). To the best of knowledge no serious, harmful effects have been reported for these doses of vitamin D.

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Gurley, Susan, David Ortiz Melo, Natalie M. Ortiz, Jessica Prescott, Katherine Xu, Robert Griffiths, and Kevin Burns. "Abstract 088: Angiotensin Converting Enzyme 2 (ACE2) in the Kidney: Soluble ACE2 Restores Blood Pressure Regulation." Hypertension 64, suppl_1 (September 2014). http://dx.doi.org/10.1161/hyp.64.suppl_1.088.

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Angiotensin converting enzyme 2 (ACE2) is a carboxypeptidase that metabolizes angiotensin II (AngII) to Ang(1-7). ACE2 can be cleaved by metalloproteinases to generate soluble ACE2 (sACE2), which remains catalytically active. The objective of this study is to determine if sACE2, acting in the kidney, could modulate the BP response to AngII infusion.Data from our kidney cross-transplant study using male (129/SvEv x C57BL/6)F 1 ACE2 KO and WT mice suggest that sACE2 can reach the urinary compartment even when not expressed in kidney cells. For these experiments, we generated 4 experimental groups: 1) WT, 2) Kidney ACE2KO, 3) Systemic ACE2KO, and 4) Total ACE2KO. Mean arterial pressures (MAP) were measured with radiotelemetry. AngII was infused chronically at 1ug/kg/min. Enzymatic activity was determined using an ACE2-specific quenched fluorescent substrate assay. ACE2 protein in the urine was confirmed by western blots using a specific N-terminus antibody. Total ACE2KO mice had an enhanced hypertensive response to AngII compared to WT (152±6 vs.144±7 mmHg; p =0.03). The presence of ACE2 in either kidney or systemic tissues restored MAPs to levels similar to WT (kidney ACE2KO 140±7mmHg, systemic ACE2 KO 140±5 mmHg, p<0.01 vs. total ACE2KO). However, Kidney ACE2KO mice had similar urinary ACE2 activity as systemic ACE2KO (76±32 and 108±52 RFU/ul/hr, respectively, p=NS). We confirmed this was ACE2 with immunoblotting of urine samples. As expected, we detected a 92kDa band in WT and systemic KO mice, which represents full length ACE2. A smaller band (70kDa) was present in the WT, systemic KO, and kidney KO groups, and absent in total KO mice. This likely represents sACE2. Moreover, the presence of the smaller band correlated with urine ACE2 activity and was associated with attenuated BPs. Our studies demonstrate that the exaggerated hypertensive phenotype seen in total ACE2 deficiency can be rescued when ACE2 is restored either in the kidney or in systemic tissues. Since the Kidney KO group demonstrated ACE2 activity and protein in the urine, we suggest that soluble ACE2 can be filtered in hypertensive renal injury and may provide an additional source of balance against upregulation of the RAS within the kidney, with the overall goal of regulating BP.

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Imanullah, Moch Najib. "URGENSI PENGATURAN WARALABA DALAM UNDANG-UNDANG." Yustisia Jurnal Hukum 1, no. 2 (May 2, 2012). http://dx.doi.org/10.20961/yustisia.v1i2.10620.

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AbstractOne of the characteristics of Fundamental Research is provide an explanation a phenomenon. The purpose of this research is to describe the phenomenon of demand for franchise regulation in Indonesia. It is a normative legal research in order to examine the principles of law, the synchronization of law, and legal history. The data used were secondary data came from the primary and secondary legal materials. Validity of data was done by triangulation of sources and sources criticism. Data were analyzed using legal interpretation. The result showed that the urgency of setting a franchise in an act is due to: 1) the content material of franchise have to regulate in an act; 2) to address the sinchronization issue with the other act; 3) to harmonize the Indonesian franchise act with the franchise act from the other countries; 4) to fullfill the justice of franchisee and international franchisor. To realize the franchise act, the Indonesian government advised to make cooperation with academics, franchise business man, association, and the public to make academic legal drafting based on academic draft from BPHN with completion in accordance with the dinamics and development of franchise business in Indonesia.<ul><li>Keywords: urgency, act, franchise. </li></ul>AbstrakSalah satu karakteristik Penelitian Fundamenatal adalah memberikan penjelasan terhadap sebuah fenomena, maka tujuan penelitian ini diarahkan untuk menjelaskan adanya fenomena permintaan pengaturan waralaba di Indonesia dalam sebuah undang-undang khusus waralaba. Untuk mencapai tujuan ini, maka dilakukan penelitian hukum normatif dalam ranah asas-asas hukum, sinkronisasi hukum, dan sejarah hukum. Data yang dipergunakan adalah data sekunder yang bersumber dari bahan hukum primer dan bahan sekunder. Kesahihan data dilakukan dengan kritik sumber. Data analisis dengan cara melakukan penafsiran hukum (gramatikal). Hasil penelitian menunjukkan bahwa urgensi pengaturan waralaba dalam sebuah undang-undang adalah karena : 1) muatan materinya harus diatur dalam undang-undang (seperti : asas-asas hukum, kewarganegaraan dan hak-haknya, kelembagaan negara, dan perpajakan); 2) untuk mengatasi persoalan sinkroniasi dengan undang-undang lain yang terkait; 3) untuk melakukan harmonisasi peraturan perundang-undangan waralaba Indonesia dengan undang-undang waralaba khusus negara lain; 4) untuk memenuhi rasa keadilan para pelaku usaha waralaba khususnya pelaku usaha waralaba internasional (asing maupun warga negara Indonesia). Untuk merealisasikan undang-undang waralaba, Pemerintah disarankan untuk bekerjasama dengan akedemisi, kalangan pengusaha waralaba, asosiasi, dan masyarakat luas untuk membuat naskah akademis undang-undang waralaba berbasis naskah akademis yang telah dihasilkan BPHN dengan penyempurnaan sesuai dengan dinamika dan perkembangan usaha waralaba di Indonesia.<ul><li>Kata kunci : urgensi, undang-undang, waralaba.</li></ul>

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Westbrook, Lindsey, Kevin R. Regner, Ashley C. Johnson, Jonathan Lee, Patrick B. Kyle, David L. Mattson, and Michael R. Garrett. "Abstract 1: Loss of Nuclear Receptor 4a1 on a Hypertensive Genetic Background Promotes Immune-Mediated Renal Injury." Hypertension 60, suppl_1 (September 2012). http://dx.doi.org/10.1161/hyp.60.suppl_1.a1.

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Nuclear hormone receptors of the NR4A subgroup have been implicated in cancer, atherosclerosis, and most prominently in metabolic disease. However, little is known about the role of NR4A1 in kidney health or disease. Fawn-hooded hypertensive (FHH) Nr4a1 -/- deficient animals were evaluated for blood pressure, proteinuria, renal function and metabolic parameters from 4-24 weeks of age. At week 24, Nr4a1 -/- rats exhibited 4-fold higher proteinuria (81±11.5 mg/24hrs/100gBW) compared to FHH (21±1.0). No difference in telemetry measured blood pressure was observed at any time point, but glomerular filtration rate was significantly decreased in the Nr4a1 -/- (542±78.9 ul/min/gKW) vs FHH (937±75.2). Moderate increases in glomerulosclerosis were seen, but were not different between strains. The severity of tubular atrophy, casts, and fibrosis was significantly increased in Nr4a1 -/- compared to FHH (19±1.86% vs 12±0.8%, respectively). Tubulointerstitial macrophage infiltration was also significantly increased in kidneys from Nr4a1 -/- (245±62 foci per field) compared to FHH (50±13.4) at week 24. Microarray analysis of kidney from week 8-24 found significant up-regulation of important immune mediators, including TGF-β1-2, CCL2 (MCP-1), CCL9 (MIP-1γ), and other chemokines and interleukins. The specific injury (tubulointerstitial), immune cell infiltration, and immune gene expression profile led to the hypothesis that loss of Nr4a1 contributes to the observed injury by an immune-mediated mechanism. To test this hypothesis, bone marrow (bm) cross transplantation studies were performed in 3 groups of irradiated (irr) animals: (1) bmFHH into irrFHH (FHH control); (2) bmFHH into irrNr4a1 -/- ; and (3) bmNr4a1 -/- into irrNr4a1 -/- (Nr4a1 control). The bmFHH into irrNr4a1 -/- animals showed a significant attenuation of proteinuria starting as early as week 12 and continuing through week 24 (35±4.6 mg/24hrs/100gBW) compared to the Nr4a1 -/- control (60±7.5). These data strongly suggest that the mechanism of Nr4a1 -/- renal injury is mediated via an immune mechanism, likely due to the predisposition of the FHH to develop renal injury. Further studies are now underway to better understand the precise immune mechanism that promotes injury in this model.

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Shoemaker, Robin C., and Lisa A. Cassis. "Abstract 63: ACE2 Deficiency Reduces Insulin Content of Isolated Pancreatic Islets and Plasma Insulin Concentrations Post-Glucose Challenge in Obese C57BL/6 Mice." Hypertension 62, suppl_1 (September 2013). http://dx.doi.org/10.1161/hyp.62.suppl_1.a63.

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Objective: Diet-induced obesity promotes type 2 diabetes (T2D). Drugs that inhibit the renin-angiotensin system (RAS) have been demonstrated in clinical trials to decrease the onset of T2D. Angiotensin converting enzyme 2 (ACE2) negatively regulates the RAS by catabolizing angiotensin II (AngII). Preliminary data indicate that ACE2 deficient mice display impairments in glucose homeostasis at 8 weeks of age. We tested the hypothesis that ACE2 deficiency promotes the development of glucose intolerance and β-cell dysfunction in mice with diet-induced obesity. Methods and Results: Male Ace2 +/y or -/y mice were fed a low fat (LF, 10% kcal as fat) or high fat (HF, 60% kcal as fat) diet for 5 or 17 weeks. After 5 weeks, plasma insulin concentrations (0, 30 min) following a glucose challenge were significantly greater in HF versus ( vs) LF-fed mice. However, glucose-stimulated increases in plasma insulin concentrations were decreased in HF-fed ACE2 deficient mice compared to controls (2.96 ± 0.18 vs 4.44 ± 0.40 ng/ul, respectively; P<0.01). Surprisingly, isolated pancreatic islets from HF-fed mice of either genotype released similar concentrations of insulin in response to glucose. However, mRNA abundance of insulin was significantly reduced in islets from HF-fed Ace2 -/y compared to +/y mice (1.76 ± 0.17 vs 2.54 ± 0.18 insulin/18S ratio; P<0.05). After 17 weeks, the plasma insulin response to glucose was further reduced in the HF-fed ACE2 deficient mice compared to controls (8.07 ± 0.98 vs 13.90 ± 1.10 ng/ul; P<0.01). Further, LF-fed ACE2 deficient mice also displayed reductions in plasma glucose-stimulated insulin concentrations (1.92 ± 0.98 vs 3.09 ± 0.98 ng/ul; P<0.01). Islets from HF-fed wild type mice displayed reduced ACE2 gene expression compared to LF (0.069 ± 0.009 vs 0.169 ± 0.01, ACE2/18S ratio; P<0.001) and AngII totally suppressed islet glucose-stimulated insulin secretion compared to vehicle (-0.16 ± 0.18 vs 0.9 ± 0.26, fold change over basal; P<0.05). Conclusions: These results demonstrate that ACE2 deficiency promotes the development of T2D by regulating islet insulin content. Moreover, diet-induced obesity reduces islet ACE2 gene expression with augmented AngII-induced impairment of insulin secretion.

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Holvik, Kristin, Livar Frøyland, Margaretha Haugen, Martinus Løvik, Tor A. Strand, Grethe S. Tell, and Per Ole Iversen. "Risk Assessment of "Other Substances" – L- tryptophan." European Journal of Nutrition & Food Safety, August 5, 2020, 75–77. http://dx.doi.org/10.9734/ejnfs/2020/v12i830268.

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The Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) has, at the request of the Norwegian Food Safety Authority (Mattilsynet; NFSA), assessed the risk of "other substances" in food supplements and energy drinks sold in Norway. VKM has assessed the risk of doses given by NFSA. These risk assessments will provide NFSA with the scientific basis for regulating the addition of "other substances" to food supplements. "Other substances" are described in the food supplement directive 2002/46/EC as substances other than vitamins or minerals that have a nutritional or physiological effect. "Other substances" are added mainly to food supplements, but also to energy drinks and other foods. VKM has not in this series of risk assessments of "other substances" evaluated any claimed beneficial effects from these substances, only possible adverse effects. The present report is a risk assessment of L-tryptophan and is based on previous risk assessments of L-tryptophan and scientific papers retrieved from systematic literature searches. L-tryptophan is an indispensable amino acid in humans, which in addition to its role in protein synthesis, also participates in complex metabolic pathways where it acts as a precursor to the potent neurotransmitter serotonin, the hormone melatonin, and the vitamin niacin (vitamin B3). L-tryptophan is available from a wide variety of protein-rich foods in the normal diet, including meat, fish, milk and dairy products, egg, beans, lentils and also bread and grains, pasta, rice, fruit and vegetables. According to information from NFSA, L-tryptophan is an ingredient in food supplements sold in Norway. NFSA has requested a risk assessment of the following doses of L-tryptophan in food supplements: 250 mg/day, 300 mg/day, and 450 mg/day for adults, adolescents and children 10 years and above. Usual dietary intake of L-tryptophan in Norway is not known, but data from the USA and the UK suggest an average dietary intake of about 900 mg/day of which the main part is bound in food protein. In phase 1 we have identified seven previous reports that have aimed to assess the safety of L-tryptophan supplementation in humans; the most recent was published by VKM in 2013. To complement the existing reports, a literature search was performed in MEDLINE and EMBASE to retrieve studies published in the period 2012-2015. This search retrieved two recent randomised trials with L-tryptophan. In addition, we performed a literature search concerning safety of L-tryptophan in children and adolescents with no time restriction. This search retrieved no relevant results that met the inclusion criteria. Four aspects related to safety of L-tryptophan were identified in previous reports: 1) adverse effects reported at high doses, including appetite suppression, nausea and vomiting, faintness, dizziness, drowsiness, tremor, fatigue, and headache; 2) a suggested, but not established, increased risk of cataract; 3) the eosinophilia-myalgia syndrome (EMS), which is thought to be caused by contaminants produced in the manufacturing of L-tryptophan supplements, however this is still unresolved; 4) the risk of adverse drug reactions caused by excessive serotonergic action by concomitant use of antidepressants, including monoamine oxidase inhibitors, selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants and other drugs, known as the serotonin syndrome. According to previous reports, doses of 3 to 6 g/day of L-tryptophan have been associated with adverse effects. Using an uncertainty factor of 10, conclusions in previous reports have suggested a maximum level of 220 mg/day for adults. An upper tolerable intake level (UL) of 220 mg/day was first proposed in a report by the UK Committee on the Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) in 2004, and was derived from the average dose of L-tryptophan consumed as a prescription drug against depression in the UK at the time (2228 mg/day). This level has been maintained in later reports by other committees, most recently VKM in 2013 as a tentative guidance level. Additional information from the publications retrieved in the literature search did not provide evidence of sufficient weight to change the previous conclusions concerning UL. There is a lack of well-designed supplementation studies with L-tryptophan in humans designed to address adverse effects and dose-response relationship as primary outcome. There is also a lack of data about potential adverse health effects of L-tryptophan supplementation in children and adolescents. Patients using antidepressant drugs constitute a specific vulnerable subgroup of the population with regard to possible adverse effects of L-tryptophan supplements, due to the potentially life-threatening drug interaction effects that occur from excessive serotonergic action. The Norwegian Scientific Committee for Food Safety (VKM) concludes that: In adults (≥18 years), the specified doses 250, 300, and 450 mg/day L-tryptophan in food supplements may represent a risk of adverse health effects. In adolescents (14 to <18 years), the specified doses 250, 300, and 450 mg/day L-tryptophan in food supplements may represent a risk of adverse health effects. In children (10 to <14 years), the specified doses 250, 300, and 450 mg/day L-tryptophan in food supplements may represent a risk of adverse health effects. Children below 10 years were not included in this assessment.

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Afzal, Dr Saira. "Endemic Crimean Congo Hemorrhagic Fever in Pakistan." Annals of King Edward Medical University 22, no. 3 (January 20, 2017). http://dx.doi.org/10.21649/akemu.v22i3.1480.

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“In times of stress and danger such as come about as the result of an epidemic, many tragic and cruel phases of human nature are brought out, as well as many brave and unselfish ones.”William Crawford Gorgas Crimean Congo hemorrhagic fever is endemic in certain parts of world. It is a <a title="Zoonotic" href="https://en.wikipedia.org/wiki/Zoonotic">zoonotic</a> disease and reservoirs are domestic and wild animals. It spreads by vector <a title="Hyalomma" href="https://en.wikipedia.org/wiki/Hyalomma">Hyalomma</a> tick or contact with infected animals or people or infected secretions. The clinical disease spectrum includes fever with flu like symptoms, hemorrhages and mortality rate of 10 – 40%. The incubation period is 1 – 3 days after a tick bite or 5 – 6 days following exposure to infected blood or tissues. The <a title="Influenza" href="https://en.wikipedia.org/wiki/Influenza">flu</a> – like symptoms may resolve after one week. In up to 75% of cases, however, signs of <a title="Hemorrhage" href="https://en.wikipedia.org/wiki/Hemorrhage">hemorrhage</a> appear within 3–5 days of the onset of illness in the form of skin bruises, nose bleeds, vomiting, and black stools. The <a title="Liver" href="https://en.wikipedia.org/wiki/Liver">liver</a> becomes swollen and tender. Patients usually begin to show signs of recovery after 9 – 10 days from when symptoms presented.1 10 – 40% of the cases result in mortality by the end of the second week of illness and may be attributed by hemorrhagic shock, hypovolemia, septacaemia, acute kidney failure, and acute respiratory distress syndrome.2 Pakistan has witnessed severe outbreaks in 2009 and 2010. In 2009, epidemic of Crimean Congo hemorrhagic fever was reported from Baluchistan. In September 2010, an outbreak was reported in Pakistan’s <a title="Khyber Pakhtunkhwa" href="https://en.wikipedia.org/wiki/Khyber_Pakhtunkhwa">Khyber Pakhtunkhwa</a> province. Poor diagnosis and record keeping has caused the extent of the epidemic to be uncertain, though some reports indicate over 100 cases, with a case – fatality rate above 10%. Crimean – Congo haemorrhagic fever is declared endemic in Pakistan. Human infections caused by the Crimean-Congo haemorrhagic virus have been occurring throughout the year and in wide geographic areas of the country. The seasonal spike has been reported this year and guidelines for prevention in public and health care providers are formulated. However, clear and rational policies from law enforcement agencies to avoid spread from endemic foci to other non-endemic areas through transportation of animals or contact with infected cases especially during Eid festivals are still needed. The transportation of animals is greatly increased during Eid festival in Pakistan and risk of epidemic is also increased. Law enforcement and Agricultural regulations require de-ticking farm animals before transportation or delivery for slaughter. Protocols for safety during slaughter and disposal of infected wastes should be formulated and implemented. In the case when feverish patients with evidence of bleeding are reported, emergency preparations for resuscitation or intensive care are required urgently. Moreover guidelines regarding suspected cases quarantine, body secretions and wastes isolation and disposal in health care facilities and standard precautions for laboratory workers, nursing staff and doctors should be adopted. Surveillance and laboratory diagnosis for early detection of cases, infection control measures in health care facilities and risk communication should be strengthened especially in high risk areas in the country. Seroprevalence of antibodies against Crimean Congo hemorrhagic virus in our community is still unknown. Preventive steps are simple but awareness in masses about Crimean congo hemorrhagic fever is the most important step. Some of the important steps for prevention are:<ul><li>Use a repellent containing 20% – 30% DEET or 20% Picaridin. Re-apply according to manufacturer’s directions.</li><li>Wear neutral – coloured and light – weight clothes, long – sleeved shirts and full pants. Tuck pants into socks for further protection.</li><li>Apply a permethrin spray or solution to clothing and gear.</li><li>When walking through grass lands avoid tall grasses and shrubs.</li><li>Carefully examine body, clothing, gear, and animals for ticks.</li><li>Apply sunscreen first followed by the repellent and preferably 20 minutes later.</li><li>Avoid coming into contact with the blood or tissues of animals. Healthcare practitioners should take appropriate infection control measures to prevent infection. Standard operating procedures to handle infectious materials and suspected cases should be displayed in clinical settings.</li><li>Laboratory staff should wear protective gear and waste disposal should be according to the protocols.</li></ul> There is no effective commercially available vaccine or chemoprophylaxis against Crimean-Congo Hemorrhagic Fever. Thus efforts should be directed to prevent this disease by awareness in masses. Moreover, seroprevalence in general public as well as in specific groups including health care providers, laboratory workers, butchers, veterans and surgeons should be detected by screening and later on confirmed by Enzyme Linked Immunosorbant Assay (ELISA). Early case detection, quarantine of susceptible cases and adoption of standard protocols during management of patients can decrease the spread of this deadly virus.

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Childhood Studies, Journal of. "Call for Papers - Innovative Professional Learning in Early Childhood Education and Care: Inspiring Hope and Action." Journal of Childhood Studies 41, no. 3 (December 22, 2016). http://dx.doi.org/10.18357/jcs.v41i3.16399.

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Guest editors: Joanne Lehrer (Université du Québec en Outaouais), Christine Massing (University of Regina), Scott Hughes (Mount Royal University), and Alaina Roach O’Keefe (University of Prince Edward Island)Not only is professional learning conceptualised as critical for increasing educational quality and enhancing children’s learning and developmental outcomes (e.g. Lazarri et al., 2013; Munton et al., 2002; Penn, 2009; Vandenbroeck et al., 2016), but specific elements of professional learning (in both initial and continuing education, or preservice and in-service learning) have been identified as essential to transforming early childhood educators’ and preschool teachers’ professional identities and practice. For example, critical and supported reflection (Thomas & Packer, 2013), learning experiences that target entire teams (Vangrieken, Dochy, & Raes, 2016), collaborative and empowering practice (Helterbran & Fennimore, 2004), and competent leadership (Colmer et al., 2008) have all been found to be effective means of supporting professional learning.While there appears to be consensus in the literature around what needs to be done, and even around how it should be done, numerous constraints prevent the implementation and maintenance of sustainable and transformational professional learning in ECEC. Vandenbroeck and colleagues (2016) go beyond the focus on individuals and childcare teams, identifying two further levels necessary for competent systems of professional learning: partnerships between local early childhood programs and social, cultural, and educational institutions (such as colleges and universities); and governance regarding vision, finance, and monitoring. In the Canadian context, the Canadian Child Care Federation has also stressed the importance of a system-wide strategy to strengthen the child care workforce (CCCF, 2016). However, early childhood services in Canada are under the purview of the provincial and territorial governments and, therefore, the conditions, regulations, certification requirements, curriculum documents, and educational systems vary widely from jurisdiction to jurisdiction. The educational requirements for certification, for example, may include no formal training (in NWT and Nunavut), one entry-level short course, one-year certificates, or two-year diplomas. This complicates efforts to define who the early childhood professional is and what opportunities are constitutive of professional learning (Prochner, Cleghorn, Kirova, & Massing, 2016). While these disparities within the field may impede the development of a cohesive strategy, Campbell et al. (2016) recently asserted that much can be learned from sharing and appreciating the rich diversity of approaches to professional learning both within and across provinces and territories. In addition, examples from other countries serve to broaden the discussion and expand our understanding of what is possible (Vandenboreock et al., 2016).This special issue, then, is dedicated to sharing stories of hope and coordinated action, linking theory with practice. We seek Canadian and international submissions related to professional learning practices that extend beyond individual programs, showcasing partnerships and community mobilization efforts within and across various settings for young children (child care, Kindergarten, drop-in centres, etc.) in relation to philosophical, practical, critical, transformative, personal, and/or hopeful themes. Each submission will respond to one or more of the key questions, including, but not limited to:<ul><li>How can professional learning be conceptualised?</li><li>How do we build and maintain effective partnerships to foster professional learning?</li><li>What strategies for transformative community mobilization might be shared?</li><li>How can innovative strategies be applied on a wider scale?</li><li>How might taken-for-granted professional learning and evaluation practice be disrupted?</li><li>What story about professional learning do you need (or want) to tell?</li><li>How has your community been transformed through a particular activity, event, or practice?</li><li>How might the lives and futures of children be positively shaped by engagement in partnerships and mobilization?</li><li>Where might we be in 5, 10, or 15 years through such endeavours?</li></ul>We welcome submissions in multiple formats, including research articles, theoretical papers, multimedia pieces, art work, book reviews, and so forth. These may be submitted in English, French, or in any Canadian Indigenous language. Submissions are due August 1, 2017 and should be submitted as per <a href="http://journals.uvic.ca/index.php/jcs/about/submissions#onlineSubmissions">Journal of Childhood Studies submission guidelines. </a> ReferencesCampbell, C., Osmond-Johnson, P., Faubert, B., Zeichner, K., Hobbs-Johnson, A. with S. Brown, P. DaCosta, A. Hales, L. Kuehn, J. Sohn, & K. Steffensen (2016). The state of educators’ professional learning in Canada. Oxford, OH: Learning Forward.Canadian Child Care Foundation [CCCF], (2016). An Early Learning and Child Care Framework for Canada’s Children. Retrieved from: http://www.cccf-fcsge.ca/wp-content/uploads/CCCF_Framework-ENG.pdfColmer, K., Waniganayake, M. & Field, L. (2014). Leading professional learning in early childhood centres: who are the educational leaders?, Australasian Journal of Early Childhood, 39(4), 103-113.Helterbran, V.R. & Fennimore, B.S. (2004). Early childhood professional development: Building from a base of teacher investigation. Early Childhood Education Journal, 31(4), 267-271.Lazarri, A., Picchio, M., & Musatti, T. (2013). Sustaining ECEC quality through continuing professional development: systemic approaches to practitioners’ professionalization in the Italian context. Early Years: An International Research Journal, 33(2), 133-145.Munton, T., Mooney, A., Moss, P., Petrie, P., Calrk, A., Woolner, J. et al., (2002). Research on ratios, group size, and staff qualifications and training in early years and childcare settings. London: University of London.Penn, H. (2009). Early childhood education and care: Key lessons from research for policy makers. Brussels: Nesse.Prochner, L., Cleghorn, A., Kirova, A., & Massing, C. (2016). Teacher education in diverse settings: Making space for intersecting worldviews. Rotterdam, The Netherlands: Sense Publishers.Thomas, S., & Packer, D. S. (2013). A Reflective Teaching Road Map for Pre-service and Novice Early Childhood Educators. International Journal of Early Childhood Special Education, 5(1), 1-14.Vandenbroeck, M., Peeters, J., Urban, M. & Lazzari, A. (2016). Introduction. In M. Vandenbroeck, M. Urban & J. Peeters (Eds.) Pathways to Professionalism in Early Childhood Education and Care, (pp. 1-14). London: Routledge.Vangrieken, K., Dochy, F., & Raes, E. (2016). Team learning in teacher teams: team entitativity as a bridge between teams-in-theory and teams-in-practice. European Journal Of Psychology Of Education - EJPE (Springer Science & Business Media B.V.), 31(3), 275-298. doi:10.1007/s10212-015-0279-0

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