A group of hematologists, involved with hemophilia research and care in the U.S.A., met under the sponsorship of the Division of Blood Diseases and Resources of the National Heart and Lung Institute. In order to improve future communication among ourselves, we decided to alter our individual methods of measurement of inhibitors to the extent necessary to permit a uniform, although arbitrary, description of inhibitor units. We agreed to the following standards: (1) The incubation mixture consists of one part citratecl patient plasma, undiluted or diluted, plus an equal part of citrated pooled normal human plasma. (2) A control incubation mixture consists of equal parts of normal pooled plasma and imidazole buffer, as formulated by Dr. Biggs. (3) The mixtures are incubated at 37° C for two hours. (4) Assays specific for Factor VIII are then performed and the Factor VIII activity in the patient mixture is divided by the Factor VIII activity in the control mixture to determine the percent residual Factor VIII activity. (5) A patient plasma giving a residual Factor VIII activity of 50 percent in this test is said to contain one “Bethesda unit” of inhibitor per ml. (6) On a graph, the log percent residual Factor VIII activity is plotted against inhibitor units. If the residual Factor VIII activity of the incubation mixture is between 75 and 25 percent, the inhibitor units are read from the graph. Plasmas containing strong inhibitors are diluted with imidazole buffer before being placed in the incubation mixture. A dilution is sought which will result in a residual Factor VIII activity between 75 and 25 percent. The units of inhibitor read from the graph are then multiplied by the dilution factor to determine the number of Bethesda units of inhibitor per ml of undiluted patient plasma.We invite interested colleagues to join us in the use of this method, and we invite discussion of better methods of describing inhibitor potency.

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The biochemical defect of cultuired skin fibroblasts from Hurler or Hunter patients (faulty degradation of sulfated mucopolysaccharide, resulting in excessive intracellular accumulation) may be corrected if cells of these two genotypes are mixed with each other or with normal cells. The effect is mediated by substances released into the medium.

Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N ¼ 5) or purified unstimulated NK cells from peripheral blood (N ¼ 4) resulted in good cell viability with eGFP expression in 85 ± 6% and 86 ± 4%, 24 h after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19 þ target cells resulting in X80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 h after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer. Cancer Gene Therapy ( IntroductionThe capacity of natural killer (NK) 1 cells to exert cytotoxicity against a variety of cancer cell types makes them an attractive tool for anti-cancer therapy. [2][3][4][5][6][7] Data gathered in the setting of allogeneic hematopoietic stem cell transplantation indicate that donor selection based on the degree of mismatch between expression of killer immunoglobulin-like receptors on donor NK cells and HLA Class I molecules expressed by the patient cells should maximize NK cell killing of target cells, 4,[8][9][10] hence augmenting the efficacy of hematopoietic stem cell transplantation. 6,7,11 In addition, it was reported that the infusion of haploidentical NK cells in a nonmyeloablative transplant setting could produce remissions in patients with acute myeloid leukemia. 5 Although NK cell cytotoxicity has a wide spectrum, some cancer cell types appear less susceptible or refractory to NK cell killing, because of failure to activate NK cells, induction of suppression or both. Among these relatively NKresistant cell types are lymphoid malignancies such as acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (B-CLL) and B-cell non-Hodgkin lymphoma. 3,[12][13][14][15][16] Chimeric antigen receptor has been studied since late 1980s. [17][18][19][20][21][22] It generally contains a single chain variable fragment as the extracellular antigen recognition unit and multiple lymphocyte activation domains as the intracellular activation part. Most work has been focused in arming T cells with this chimeric antigen receptor for antitumor effect 21-23 NK cells transduced with chimeric antigen receptor have also been exploited for anti-tumor effect. Various com...