ALBERT

Description: Journal of Medicinal Chemistry DOI: 10.1021/jm400645w

Description: Journal of Medicinal Chemistry DOI: 10.1021/jm301550c

Description: Journal of Medicinal Chemistry DOI: 10.1021/jm3010459

Description: Stromal cells in the bone marrow provide an optimal microenvironment for hematopoiesis through cell-to-cell interaction as well as cytokine production. We have previously established 33 bone marrow stromal cell lines from SV40 T-antigen transgenic mice, and demonstrated the lineage-specific activity of supporting hematopoiesis (J Cell Physiol1995: 164; 55). Of the 33 cell lines, 27 clones showed stimulatory abilities to support large erythroid colony formation in the presence of erythropoietin, while 6 cell lines did not. Since these activities were not dependent on the amount of produced cytokines, the existence of unknown molecules responsible for the selective activity of erythropoiesis has been implicated. To identify molecules expressed on bone marrow stromal cells which are responsible for determining the stromal cell dependent erythropoiesis, we compared the gene expression profiling by cDNA microarray between cell lines which support erythropoiesis (E+; TBR 9, 184, 31-2) and cell lines which do not (E-; TBR 17, 33, 511). Out of 7226 genes examined, 138 genes were up-regulated 3-fold or more, and 282 genes were down-regulated 3-fold or more, in E+ cell lines. Among these genes, we have selected one of the up-regulated genes, tenascin-C, as a candidate for erythroid progenitor supporting genes, and examined its expression and function in detail. First, we confirmed the expression of tenascin-C by real time quantitative RT-PCR. The expression of tenascin-C adjusted by the expression of GAPDH in the three E+ cell lines were all higher than the three E- cell lines with a mean of 3.6 times. Next, we examined the effect of suppressing tenascin-C expression by small interfering RNA (siRNA) on the erythroid colony formation. siRNAs were synthesized by in vitro transcription method, and were transfected into TBR cell lines by lipofection. At 24 hours after the transfection of tenascin-C siRNA, the expression of tenascin-C was reduced to 10 to 40% of the control siRNA as determined by real time quantitative RT-PCR. The number of erythroid colony dependent on TBR 9 and TBR 184 in the presence of tenascin-C siRNA was significantly lower than that in the presence of control siRNA (13.0±6.5 versus 0.75±0.95 colonies/104 fetal liver cells; p

Description: Background: Thrombotic microangiopathy (TMA) and sinusoidal obstruction syndrome (SOS) as well as acute GVHD are major complications after allogeneic hematopoietic stem cell transplantation (alloHSCT) to overcome to achieve better overall survival. These complications are closely relevant to inflammation and coagulation involving endothelium, which are named as transplantation-associated coagulopathy (TAC). Recombinant thrombomodulin (rTM) is a new drug for treating DIC approved by Japanese Ministry of Health, Labour and Welfare in 2008. Membranous TM has many aspects to inhibit inflammation via activated Protein C (APC), lectin-like domain, and activated thrombin-activatable fibrinolysis inhibitor as well as to inhibit coagulation by binding factor IXa and Xa through APC. Biomarkers such as inflammatory cytokines and endothelial molecules are expected to be clue in elucidating TAC. We established SIGHT study group to explore above complications following alloHSCT. In the present study, we investigated the effects of rTM on levels of the biomarkers and whether we could prevent TAC using rTM among registered patients in the SIGHT study group. Patients and methods: SIGHT study group covered nationwide institutes more than 70 in Japan. Patients whoever were eligible for alloHSCT to treat hematopoietic disorders by physicians decision were permitted and written informed consent was requested to register in the study. Blood samples were collected from the patients before and after transplantation through day 28. Levels of cytokines (IL-6, TNF-α, HMGB1, MCP-1, RANTES) and soluble molecules (VCAM-1, E-selectin, PAI-1, PDMP) were measured by ELISA. rTM was administered as a prophylactic therapy for TAC in day 4-14 after alloHSCT. Control group was received heparin or no anti-coagulation therapy as prophylaxis during same schedule as rTM group. Patients were not randomized to these three arms but allowed to select one by their physicians decision. The primary endpoint was a comparison of fluctuation of these biomarkers in three arms. Secondary endopoint was a comparison of the rate of developing TAC. Results: The subjects were 293 patients who underwent alloHSCT in SIGHT study group between June 2010 and February 2014. 271 patients out of them were analyzed to measure level of these biomarkers. There was no significant difference in all characteristics but GVHD prophylaxis between two groups. rTM group received more cyclosporine than control group (p=0.05). MCP-1 and IL-6 exhibited more significant elevations on day 7 after alloHSCT, while HMGB-1 did on the day of alloHSCT. In contrast, the levels of TNF-α, E-selectin, VCAM-1, PAI-1 and PDMP exhibited increment since around day 7 after alloHSCT. Significant improvements in TNF-α, E-selectin, VCAM-1, HMGB-1, PAI-1 and PDMP was shown after rTM-treatment, but not shown in control group. Regarding complications following alloHSCT, the rate of developing acute GVHD and SOS was significantly lower in rTM group than without it (36.5% vs 62.2%, p=0.000; 12.5% vs 24.5%, p=0.032, respectively), while TMA did not differ (8.4% vs 10.2%, p=0.961). Stepwise multivariate logistic regression analyses revealed that anti-coagulation therapy without rTM and the level of PAI-1 on day 28 after HSCT were independent risk factor for acute GVHD (p=0.000, odds ratio=3.006; p=0.000, odds ratio=1.068, respectively). Also anti-coagulation therapy without rTM and the level of HMGB-1 on day 28 after HSCT were significantly predictive for risk of SOS (p=0.015, odds ratio=2.650; p=0.001, odds ratio=1.433, respectively). Tacrolimus was significantly good risk of SOS (p=0.022, odds ratio=0.404). The level of VCAM-1 on day 28 after HSCT was significantly associated with risk of TMA (p=0.040, odds ratio=1.001). Conclusions: We identified predictive biomarkers for TAC following alloHSCT, which were PAI-1 for acute GVHD, HMGB-1 for SOS, and VCAM-1 for TMA, respectively in this study. The present findings suggest that rTM has the possibility to play a therapeutic role for acute GVHD and SOS induced by suppression on these biomarkers. Endothelial and pro-coagulant biomarkers were released delayed compared with inflammatory biomarkers. TMA is reported to develop around day 44 in median after alloHSCT. Further studies are needed whether rTM should administer later or longer to improve TMA than the present duration after alloHSCT. Disclosures No relevant conflicts of interest to declare.

Description: Background Levels of cytokines, chemokines and soluble molecules fluctuate after allogeneic hematopoietic stem cell transplantation (HSCT). These biomarkers are possible to be a diagnostic and prognostic value for transplantation-associated coagulopathy (TAC). In the present study, we investigated the effects of recombinant thrombomodulin (rTM) on levels of the cytokines, chemokines, and soluble factors registered in the ‘SIGHT’ research. SIGHT comprises the capital letters of five complications after HSCT, namely sinusoidal obstruction syndrome (SOS same as VOD), infection, GVHD, hemophagocytic syndrome and thrombotic microangiopathy. Methods The subjects were 159 patients who underwent allogeneic HSCT (bone marrow = 83, peripheral blood stem cells = 31, cord blood = 45). Blood samples were collected before and after transplantation. Levels of cytokines (interleukin-6, tumor necrosis factor-α, high-mobility group box (HMGB) 1), chemokines (monocyte chemotactic protein (MCP)-1, RANTES), and soluble molecules (soluble vascular cell adhesion molecule (VCAM)-1, soluble E-selectin, plasminogen activator inhibitor-1, platelet-derived microparticles (PDMP)) were measured by enzyme-linked immunosorbent assay. The rTM was administered as a therapy for transplantation-associated coagulopathy (TAC). This protocol was completed in day 4-14 after HSCT and consisted of day doses of 380 unit/kg with every days. Control group was also used heparin or no anti-coagulation therapy. Results MCP-1, IL-6, and TNF-a exhibited more significant elevations on days 7–14 after HSCT. In contrast, the levels of HMGB1, sE-selectin, sVCAM-1, PAI-1 and PDMP exhibited significant changes on days 14–28. There were significant improvements in TNF-a, sE-selectin, sVCAM-1, HMGB1, PAI-1 and PDMP after rTM-treatment (n=73), but not after rTM-untreatment patients (n=86). Conclusion We believe one of causes for TAC is pro-inflammatory cytokine including HMGB1. For this reason, it is thought that the direct anti-inflammatory effect of rTM’s lectin domein plays an important role in therapeutic mechanism for TAC. The present findings suggest the possibility that rTM can play a therapeutic role for TAC after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Backgrounds Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of T-cell lymphoma, characterized by generalized lymphadenopathy and frequent autoimmune-like manifestations. The diagnosis of AITL is sometimes challenging for hematopathologists, because the tumor cell content is generally low and relatively large reactive lymphocytes are confused as tumor cells. Possibly because of the low tumor cell frequency, clonal rearrangement of T-cell receptor gene is undetectable in 30% of the cases. We identified recurrent mutations in RHOA at c.G50T, predicting to generate p.G17V in 70% of AITL and PTCL-NOS harboring AITL features, which implies diagnostic properties for AITL (M S-Y and SC, unpublished). Purpose To establish a novel cost-effective method to diagnose AITL, we performed allele-specific realtime PCR to detect RHOA G17V mutation. Methods Genomic DNA was extracted from 119 AITL and PTCL-NOS samples, which include 47 periodate-lysine-paraformaldehyde (PLP)-fixed, 12 formalin-fixed-paraffin- embedded (FFPE) and 60 frozen tissues. Forty-one out of 60 genomic DNA samples, purified from frozen tissue, were amplified by RepliG kit (QIAGEN). Allele-specific primers for RHOA G17V mutant and wild-type sequences were designed by Wangkumhang's algorithm. The [mut] and [WT] values were individually measured by realtime PCR using each primer set, and the [mut]/([mut]+[WT]) values were calculated. Mutant allele frequencies were determined by amplicon-based deep sequencing using MiSeq. Results The [mut] values were distributed from 1.5×10-7 to 7.6×10-2, and the [WT] values were from 7.9×10-5 to 1.3×10-2. The [mut]/[mut]+[WT] values were distributed from 1.9×10-4 to 8.5×10-1. We set a cut-off value to determine existence of mutation as 2% for MiSeq, and 1.3×10-2 for the [mut]/([mut]+[WT]) value. Then we compared these two methods to detect RHOA G17V mutation. Forty-three cases were positive for the RHOA mutation in this study cohort by MiSeq, including 32 AITL and 11 PTCL-NOS cases. The [mut]/([mut]+[WT]) values of DNA from FFPE samples tend to be lower than those from other samples, and 4 out of 12 FFPE samples determined as mutation-positive by MiSeq were not detected by our allele-specific realtime PCR. We therefore excluded the FFPE samples and analyzed the data of 107 DNA samples, purified from PLP-fixed and frozen tissues. Rank correlation coefficient was 0.753. Sensitivity was 97.4%, and specificity was 97.1%. Positive concordance rate was 94.9%, and negative concordance rate was 98.6%. Conclusions We established a method to detect RHOA G17V hotspot mutation for AITL. It is expected to be highly accurate and cost effective. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Thrombotic microangiopathy (TMA) and veno-occlusive disease (VOD) as well as acute GVHD (aGVHD) are closely relevant to inflammation and coagulation involving endothelium in the setting of allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is named as transplantation associated coagulopathy (TAC) to overcome challenges and achieve better survival. Recombinant thrombomodulin (rTM) is a new drug for treating disseminated intravascular coagulation (DIC) approved by the Japanese Ministry of Health, Labour and Welfare in 2008. Membranous TM has many aspects to inhibit inflammation via activated Protein C (APC), lectin-like domain, and activated thrombin-activatable fibrinolysis inhibitor as well as to inhibit coagulation by binding factor IXa and Xa through APC. We investigated the effects of rTM on levels of the biomarkers and examined whether we could prevent TAC using rTM among registered patients in the SIGHT study group. We reported that significant improvements in elevation of TNF-α, E-selectin, VCAM-1, HMGB-1, PAI-1 and PDMP were exhibited following alloHSCT with rTM-treatment. Here we studied which biomarker were predictive for TAC and contributed to reduce the probability of these complications. Methods: SIGHT study group was composed of 70 hospitals and research centers in Japan. Patients eligible for alloHSCT treatment for hematopoietic disorders who secured attending physicians clearance were permitted in the study. Blood samples were collected from the patients before and after transplantation through day 28. Levels of cytokines (IL-6, TNF-α, HMGB1, MCP-1, RANTES) and soluble molecules (VCAM-1, E-selectin, PAI-1, PDMP) were measured by ELISA. rTM was administered as a prophylactic therapy for TAC in day 4-14 after alloHSCT. The dose of rTM, 380 u/kg, was employed as same dose as the treatment for DIC. Control group received 100 u/kg heparin, or no anti-coagulation therapy as prophylaxis at the same schedule as the rTM group. Patients were not randomized to these three arms but allowed to select one by their physician's decision. Complications following transplantation were reported by attending physicians. Results: The subjects were 293 patients who underwent alloHSCT in SIGHT study group between June 2010 and February 2014. 194 patients out of them, in whom complications following alloHSCT were reported, were analyzed to measure level of above biomarkers. No G3/4 adverse events of bleedings or severe organ damages were reported. Regarding complications following alloHSCT, the rate of developing aGVHD and VOD was significantly lower in rTM group than without it (36.5% vs. 62.2%, p=0.000; 12.5% vs. 24.5%, p=0.032, respectively), while TMA did not differ (8.4% vs. 10.2%, p=0.961). Stepwise multivariate logistic regression analyses revealed that anti-coagulation therapy without rTM was an independent risk factor for aGVHD (p=0.000, odds ratio=3.006) and VOD (p=0.015, odds ratio=2.65). We explored candidates for predictive biomarkers for TAC following alloHSCT. Only HMGB-1 levels were useful markers associated with risk of VOD among them (p=0.001, odds ratio=1.433). Also, the levels of HMGB-1 since the day of transplantation were significantly associated with risk of VOD after analyzing the whole dynamics of HMGB-1 level. E-selectin, PAI-1, VCAM-1, PDMP, and HMGB-1 were found to be closely relevant to one another by the principal component analysis (principal component loading=0.797, 0.876, 0.675, 0.780, and 0.691, respectively). Conclusion: The present findings suggested that rTM had the possibility to play a therapeutic role for aGVHD and VOD. HMGB-1 worked as an inflammatory cytokine, which exhibited peak level on the day of transplantation influenced by toxicity of conditioning regimen. VOD develop in relatively early days up to 20 days after alloHSCT, the elevation of HMGB-1 levels since day 0 after transplantation could be an early predictive biomarker for it. Once condition regimens are initiated, HMGB-1 is released from the injured cells including endothelial cells. HMGB-1 induces to release inflammatory cytokines and express adhesion molecules as well as tissue factor on endothelium (Yang H. Proc Natl Acad Sci USA. 2010;107:11942). These reactions facilitate pro-coagulant state resulting in VOD. We concluded that HMGB-1 was a major factor for VOD and rTM was effective to treat VOD attributable to suppression on HMGB-1. Disclosures Ishida: Bristol-Myers Squibb: Honoraria.