Demonstration of wideband metal mesh filters for submillimeter astrophysics using flexible printed circuits.

We developed a wideband quasi-optical band-pass filter covering 170-520 GHz by exploiting the recent advancements in commercially available flexible printed circuit (FPC) fabrication technologies. We designed and fabricated a three-layered stack of loaded hexagonal grid metal meshes using a copper pattern with a narrowest linewidth of 50µm on a polyimide substrate. The measured frequency passband shape was successfully reproduced through a numerical simulation using a set of parameters consistent with the dimensions of the fabricated metal meshes. FPC-based metal mesh filters will provide a new pathway toward the on-demand development of millimeter/submillimeter-wave quasi-optical filters at low cost and with a short turnaround time.

A new disulfide-linked dimer of a single-chain antibody fragment against human CD47 induces apoptosis in lymphoid malignant cells via the hypoxia inducible factor-1α pathway.

CD47 belongs to the immunoglobulin superfamily and is associated with ß-integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B-chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide-stabilized dimer of a single-chain antibody fragment of MABL (S-S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47-mediated apoptosis in human lymphoid malignant cells. Treatment with S-S diabody alone induced apoptosis of CD47-positive primary B-CLL and leukemic cells (MOLT-4 and JOK-1). In addition, administration of S-S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK-1 cells. In gene expression profiling of the S-S diabody-treated MOLT-4 cells, hypoxia inducible factor (HIF)-1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF-1α, RTP801 and BNIP3 were increased. Knockdown of HIF-1α by siRNA repressed S-S diabody-induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S-S diabody might have potential as a novel therapeutic agent for B-CLL.

Spatial scale and density dependence in a host parasitoid system: an arboreal ant, Azteca instabilis, and its Pseudacteon phorid parasitoid.

Much literature is dedicated to the study of density dependence in host-parasitoid systems. The theoretical literature identifies two potential stabilization mechanisms relating the response of the parasitoid to host density. One is a behavioral response that occurs at a local level, and the other is a demographic response that occurs at a larger spatial scale with heterogeneous patches of host concentrations. In a series of field trials at the small spatial scale (tens of meters) and a 10-mo census at the large spatial scale (hundreds of meters), we examined the attack rates of Pseudacteon sp. phorid flies on Azteca instabilis F. Smith ant hosts. At the local scale, we examined phorid attack rates on ants placed at increasing distances from ant nests (0-12 m) and on different densities of ants (1-50 workers) placed near the nests. At the large scale, we placed whole body extracts of A. instabilis in areas with several A. instabilis nests and in areas >100 m from the nearest nest. For all observations, we recorded the time of the first phorid attack and the number of phorid attacks in a defined time period. We found clear density-dependent responses at both scales. Phorid attack rates were highest within 2 m of A. instabilis nests and at ant densities >25. At the larger scale, phorid attack rate was greater in areas with A. instabilis nests, although this result was obscured during the dry season when the population of parasitoids is dramatically reduced. We propose several behavioral and population-level responses that may contribute to the observed results and propose several testable hypotheses. We conclude that, in this system, density dependence does happen through a behavioral mechanism of the phorid actively seeking concentrations of ants, but also, a population level response is likely caused by the significant difference in phorid attack rates in areas far from ant nests compared with areas nearby to nests.

Antitumor activity of a monoclonal antibody against CD47 in xenograft models of human leukemia.

The ligation of CD47 induces the apoptosis of leukemic cells in a caspase-independent manner. We generated a monoclonal antibody against CD47 (mAb-MABL) that possibly induced apoptosis from the ligation of CD47 in CCRF-CEM and JOK-1 cells in vitro. To confirm whether the ligation of CD47 caused cell death in vivo, we examined the antitumor activity of F(ab')2 of mAb-MABL in two xenograft models: The acute lymphoblastic leukemia (CCRF-CEM) and the B-cell chronic lymphocytic leukemia (JOK-1) cell line. Furthermore, in order to clarify the apoptotic activity selective for the tumor cells, we examined F(ab')2 of mAb-MABL apoptotic effects on CD34+ hematopoietic progenitor/stem and human endothelial cells. Male SCID mice were intravenously injected with CCRF-CEM (5 x 10(6) cells/mouse) or JOK-1 cells (5 x 10(6) cells/mouse) and intraperitoneally with JOK-1 cells (2 x 10(7) cells/mice). After the implantation of the cells, the mice were intravenously administered the vehicle or the F(ab')2 fragment of mAb-MABL at several doses and the length of survival was measured. F(ab')2 of mAb-MABL markedly prolonged the survival of mice transplanted with CCRF-CEM and JOK-1. Significantly, 40% of the mice intraperitoneally injected with JOK-1 cells became tumor-free when administered F(ab')2 of mAb-MABL, whereas even a high dose of fludarabine only slightly prolonged the median survival time. On the contrary, F(ab')2 of mAb-MABL showed no apoptotic effect on CD34+ hematopoietic progenitor/stem or human endothelial cells. Thus, monoclonal antibodies that cause cell death from the ligation of CD47 could be novel therapeutic agents for incurable leukemia after further optimization such as humanization or making single chain diabodies.

Apoptosis inducing bivalent single-chain antibody fragments against CD47 showed antitumor potency for multiple myeloma.

Multiple myeloma is currently considered incurable despite the use of high-dose chemotherapy with autologous hematopoietic stem cell transplantation support. Here, we show antitumor efficacy of a novel bivalent single-chain antibody fragment (scFv) against CD47 in an in vivo myeloma model. We generated two types of novel scFv molecules against CD47 having apoptosis-inducing activity for leukemic cell lines: a non-covalently linked scFv dimer (diabody) and a covalently linked bivalent scFv. Administration of these bivalent scFvs significantly prolonged the survival of mice transplanted with KPMM2 human myeloma cells. Because bivalent scFvs induced neither ADCC nor CDC, such antitumor activity by bivalent scFv is presumably attributable to cell death caused by the ligation of CD47. Thus, these apoptosis-inducing scFvs will be effective as a novel therapy for multiple myeloma which is considered incurable with conventional therapy.

Determination of concentration and binding affinity of antibody fragments by use of surface plasmon resonance.

An assay method using a surface plasmon resonance (SPR) biosensor has been developed that allows quantitative measurement of the specific antibody concentration in crude materials. By injecting non-labeled antibody samples onto a biosensor surface on which antigen was immobilized at high densities, the concentration of active antibodies can be accurately measured. To clarify applicability of this method to pharmacokinetic studies, the concentration of active antibodies in mouse plasma was measured for 4 h after injection of antibodies in mice. Although this period of measurement might be insufficient for determining the pharmacokinetics of blood pool clearance, this method has some advantages over conventional methods in measurement of single-chain antibody fragment (scFv) concentrations. Using the SPR biosensor, scFv and antibodies without epitope tag peptides were easily detected in real time, requiring as little as 20 mul of blood sample. Moreover, from the apparent dissociation rate in the dissociation phase of the sensorgrams, we could identify whether the antibody fragments existed as bivalent or monovalent in animal blood. We also evaluated the antigen binding activity of the scFvs against human CD47 and found scFvs had slightly weak affinity to their antigen (K(D), about 10 nM) compared with F(ab')2 and Fab' fragments (K(D), about 3-4 nM). This assay method promises to be a convenient tool for quality control, screening, and simple pharmacokinetic analysis of antibody fragments and other recombinant proteins not having epitope tags.

A bivalent single-chain Fv fragment against CD47 induces apoptosis for leukemic cells.

We constructed a single-chain antibody fragment (scFv) of murine monoclonal antibody, MABL, which specifically bound to human CD47 (hCD47) and induced apoptosis of the leukemic cells. The scFv of MABL antibody with a 15-residue linker (MABL scFv-15) formed both dimer (Mr 50 kDa) and monomer (Mr 25 kDa). Both MABL scFv-15 dimer and monomer had binding activity for hCD47. MABL scFv-15 dimer strongly induced apoptosis of hCD47-introduced mouse leukemic cells in vitro and exhibited anti-tumor effect in a myeloma transplanted mice model. However, MABL scFv-15 monomer scarcely exhibited these activities. These results strongly demonstrate that the ligation of CD47 antigen by two antigen-binding sites of MABL dimer is needed for inducing apoptosis. The parent MABL antibody caused hemagglutination due to the CD47 expressed on erythrocytes. Interestingly, MABL scFv-15 dimer did not cause hemagglutination. This apoptosis-inducing dimer appears to be a lead candidate for novel leukemic therapy.