[Optimization of ATG dose in haploid hematopoietic stem cell transplantation for hematologic malignancies].

Objective: To compare the clinical efficacy of different doses of rabbit antithymocyte globulin (rATG) in haplo-HSCT in the treatment of hematologic malignancies. Methods: Malignant hematological patients treated at our hospital from March 2013 to December 2018 were retrospectively analyzed. These patients were divided into three groups as per three doses of ATG (6 mg/kg, 7.5 mg/kg, and 9 mg/kg) in the conditioning regimens. The transplant outcomes were compared in terms of the occurrence of acute graft versus host disease (GVHD) , infection, and survival. Results: ①Total 288 patients were enrolled in the study, including 182 men and 106 women, with a median age of 18 (6-62) years. Total 110 patients were diagnosed with acute lymphoblastic leukemia (ALL) , 128 with acute myelogenous leukemia (AML) , 8 with chronic myeloid leukemia (CML) , 28 with myelodysplastic syndrome (MDS) , and 14 with mixed cell leukemia (MAL) . There were 159 patients in the ATG-6 group, 72 in the ATG-7.5 group, and 57 in the ATG-9 group. The median follow-up time of post transplantation was 14 (0.2-74) months. ②The incidence of neutrophil engraftment (96.9% , 97.2% , and 96.5% , respectively) and platelet engraftment (92.5% , 87.5% , and 86% , respectively) did not significantly differ among the ATG-6, ATG-7.5, and ATG-9 groups (P=0.972, P=0.276) . The incidence of grades 2-4 acute GVHD was 14.5% , 11.1% , and 8.8% in the three groups, respectively (P=0.493) , chronic GVHD incidence in the three group was 8.8% , 14.3% and 12.0% , respectively (P=0.493) . The infection rates of CMV and EBV in the ATG-9 group (77.2% and 12.5% ) were significantly higher than those in the ATG-6 (43.3% and 3.5% ) , and ATG -7.5 group (44.4% and 1.5% ) (P<0.001 and P=0.033, respectively) . ③Among the three groups, there were no significant difference in the 3-year overall survival [68.5% (95% CI 60.3% -77.9% ) , 60.1% (95% CI 48.3% -74.8% ) , 64.7% (95% CI 51.9% -80.7% ) ], cumulative incidences of relapse [34.6% (95% CI 34.3% -35.1% ) , 38.0% (95% CI 37.3% -38.7% ) , 20.6% (95% CI 20.0% -21.3% ) ], disease-free survival [53.3% (95% CI 44.9% -63.4% ) , 51.9% (95% CI 41% -65.8% ) , 63.9% (95% CI 51.9% -78.7% ) ] and non-relapse mortality [24.2% (95% CI 23.8% -24.5% ) , 26.0% (95% CI 25.4% -26.6% ) , 23.6% (95% CI 26.3% -28.2% ) ] (P=0.648, P=0.165, and P=0.486 and P=0.955) . Conclusion: Low dose (6 mg/kg) of rATG may increase the risk of grade Ⅱ-Ⅳ aGVHD, and a high dose (9 mg/kg) of ATG could significantly increase the risk of CMV and EBV infection. Median dose (7.5 mg/kg) of ATG is expected to reduce the incidence of moderate to severe aGVHD and viral infections without increasing the mortality.

Idarubicin-intensified haploidentical HSCT with GvHD prophylaxis of ATG and basiliximab provides comparable results to sibling donors in high-risk acute leukemia.

We designed a novel haploidentical hematopoietic stem cell transplantation (haplo-HSCT) system using idarubicin (IDA) intensified conditioning regimens and combination of antithymocyte globulin and basiliximab for GvHD prophylaxis. The outcomes of 110 high-risk acute leukemia patients undergoing haplo-HSCT were compared with 69 contemporaneous high-risk patients receiving HLA-matched sibling transplantation using uniform IDA-intensified regimens. The relapse incidence of haplo-HSCT was 23.4%, and 3-year overall survival (OS) and disease-free survival (DFS) achieved 62.9%, 59.1%, respectively. The cumulative incidences of II-IV and III-IV aGvHD were 28.6 and 14.3%, while limited and extensive cGvHD were 19.4, 13.8%. All these results were equivalent to those of concurrent identical sibling transplantation. Three-year OS and DFS for patients in advance stage reached 48.5, 47.3%. Furthermore, the relapse, 3-year OS of positive minimal residual disease (MRD) patients did not differ from negative MRD patients (18.9% vs 11.5%, 63.6% vs 69.6%), indicating our intensified haplo-HSCT technique could circumvent the dismal prognosis of MRD. These data provide reinforcing evidence that our haplo-HSCT system could dramatically improve the survival of high-risk acute leukemia with low relapse and acceptable transplantation-related mortality, and might be a promising therapeutic option for high-risk patients.

Phase II randomized, double-blind, placebo-controlled study of AMG 386 (trebananib) in combination with cisplatin and capecitabine in patients with metastatic gastro-oesophageal cancer.

BACKGROUND: We evaluated AMG 386, an investigational peptibody that neutralizes the interaction between angiopoietins-1 and -2 and the Tie2 receptor, combined with cisplatin/capecitabine (CX) as first-line treatment for metastatic gastro-oesophageal cancer. PATIENTS AND METHODS: Patients with metastatic gastric, gastro-oesophageal junction, or distal oesophageal adenocarcinoma were randomized 1:1:1 to CX (cisplatin 80 mg/m(2) IV Q3W; capecitabine 1000 mg/m(2) P.O. BID for 14 days Q3W) plus intravenous AMG 386 10 mg/kg QW (Arm A) or 3 mg/kg QW (Arm B), or placebo QW (Arm C). The primary end point was estimated progression-free survival (PFS). RESULTS: A total of 171 patients were enrolled. Median estimated PFS in Arms A, B, and C was 4.2, 4.9, and 5.2 months, respectively (hazard ratio for Arms A+B combined versus Arm C, 0.98; 95% CI 0.67-1.43; P = 0.92). Objective response rates were 27% (Arm A), 43% (Arm B), and 35% (Arm C). Incidence of grade ≥3 adverse events was 80% in Arm A, 84% in Arm B, and 75% in Arm C. There was no evidence of pharmacokinetic interactions. CONCLUSIONS: In this study, PFS and ORR were estimated to be similar with AMG 386 plus CX and placebo plus CX treatment. Compared with placebo, toxicity of AMG 386 plus CX was greater but manageable. PREVIOUS PRESENTATION: The results of this study have not been previously published or submitted for publication elsewhere. The results were presented in part at the Gastrointestinal Cancers Symposium, San Francisco, CA, January 20-22, 2011. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov registration number: NCT00583674.

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1.

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G(1)/S checkpoint and maintain high levels of p27(KIP1) mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27(KIP1) is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin alpha(2)beta(1), which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.

NF-Y and Sp1 cooperate for the transcriptional activation and cAMP response of human tissue inhibitor of metalloproteinases-2.

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is a key determinant in the homeostasis of the extracellular matrix. We have identified two cis-acting elements involved in the transcriptional regulation of TIMP-2. The first is an inverted CCAAT box located at position -73 to -69 in the TIMP-2 promoter that binds the transcription factor NF-Y. The second is a GAGGAGGGGG motif located at position -107 to -98, that binds the transcription factors Sp1 and Sp3. NF-Y and Sp1 cooperate for the basal transcription activity of the promoter. We then determined that TIMP-2 is transcriptionally up-regulated by cAMP analogs. Up-regulation of TIMP-2 by dibutyryl cAMP is a delayed response that requires de novo protein synthesis and does not affect RNA stability. The NF-Y and the Sp1 binding site are both involved in cAMP-dependent up-regulation of TIMP-2. Whereas NF-Y is essential for cAMP mediated regulation, Sp1 alone is not sufficient but enhances the activity of NF-Y. Dibutyryl cAMP has no effect on the expression of MMP-2 and MMP-9 and switches the MMP-TIMP balance in favor of the inhibitor.

Uptake of dopamine by rat hepatocytes in vitro.

The present results showed that uptake of dopamine (DA) by rat isolated hepatocytes was mediated, in addition to simple diffusion, mainly by a transporter-involved process, with Km of 66.8 mumol and Vmax of 52.3 pmol.min-1/10(5) cells. The process was pH- and temperature-dependent and required an activation energy of 4.12 kcal.mol-1 (Q10 = 1.25) in the range of 2.0-12.7 C and 13.0 kcal.mol-1 (Q10 = 2.0) in the range of 12.7-39.0 C. Cysteine residue having free thiol group was unrelated to the activity of the transporter. Catecholamines, serotonin, and cocaine inhibited the DA transport, but tyramine (TA) and tryptamine, as well as benztropine and imipramine (which are potent inhibitors for hepatic TA transporter and neuronal DA transporter), had no inhibitory effect on the transport of DA in these cells. These results indicated that DA was taken up into hepatocytes by a distinct carrier. NaF and mastoparan influenced the transport activity in these cells further, suggesting that signal transducing G-proteins may be involved in the regulation of DA transporter in rat hepatocytes.

Uptake by rat liver of bovine growth hormone free or bound to a monoclonal antibody.

In the work reported here, we have compared the elimination from the blood, the uptake by the liver and the intracellular distribution of bovine growth hormone, free(Gh) or bound to a monoclonal antibody (GhAb). Results show that: a) the elimination from the blood is more rapid for Gh than for GhAb; b) both molecules are quickly taken up by the liver; c) probably after travelling through endosomes, Gh and GhAb get to lysosomes where they are degraded. However, Gh mostly ends in hepatocyte lysosomes while GhAb is recovered to a large extent in sinusoidal cell lysosomes; and d) binding by isolated hepatocytes is markedly less efficient for GhAb than for Gh.

Uptake of tyramine by rat hepatocytes.

Observations on the uptake of tyramine by hepatocytes indicate that the amine is taken up by simple diffusion and a transporter mediated system, with a Km of 39 microM and a Vmax of 270 pmol/min/10(5) cells. The carrier-mediated process is pH- and temperature-dependent and requires an activation energy of 12.9 kcal/mol. An overshoot uptake is achieved a few minutes after adding this amine to the cell suspension, suggesting that active transport is involved. This is supported by the finding that partial inhibition of the uptake can be induced by oligomycin, azide, cyanide and dinitrophenol. NO3-, SCN- and SO4(2-), which change the membrane potential significantly, and depress the transporter mediated uptake further, suggesting that the membrane potential is the driving force for the entry of this amine across hepatic membrane. Cysteine is essential for the normal carrier function; whereas, histidine, tryptophan, arginine and lysine do not directly deal with the activity of the carrier. Many substances, but not amino acids, H, M, and N receptor agonists, can inhibit the uptake of tyramine. It is possible that other amines can enter hepatocytes by using this transporter.

Uptake of tyramine cellobiose by rat liver.

The uptake of 125I-tyramine cellobiose (TC) by isolated rat hepatocytes and by total rat liver is markedly higher than that of 14C-sucrose and 125I-PVP, suggesting that TC does not enter the cells by fluid phase endocytosis. The distribution of radioactivity after differential centrifugation shows that the compound is shared out amongst sedimentable structures and unsedimentable fraction. Analysis by isopycnic centrifugation indicates that quickly after its penetration into the cells, most of sedimentable 125I-TC is associated with lysosomes. Such an intracellular localization is confirmed by the distributions observed after free flow electrophoresis and by the fact that radioactivity and cathepsin C, a lysosomal hydrolase, are simultaneously released from a mitochondrial fraction treated with glycyl-L-phenylalanine-2-naphthylamide. Pretreatment of the rats with chloroquine, an acidotropic drug that accumulates in lysosomes, prevents to some extent the entry of 125I-TC into these organelles. Experiments performed with purified lysosomes show that 14C-sucrose does not cross the lysosomal membrane when 125I-TC accumulates linearly with time in the fractions. These results are explained by supposing that the linkage of tyramine to cellobiose allow the disaccharide to diffuse through the plasma and the lysosome membranes, and that the accumulation of the molecule in these organelles results from its weak basic properties. 125I-TC could be an interesting molecule with which to study acidotropism in the whole animal and in isolated and cultured cells.