A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells

Purpose In the field of cancer immunotherapy research, the targeting of effector cells with specific antibodies is a very promising approach. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems.Methods We have produced an anti-epidermal growth-factor receptor (EGFR) × anti-CD3 bispecific diabody (Ex3 diabody) in an Escherichia coli (E. coli) expression system with refolding method. The Ex3 diabody targets lymphokine-activated killer cells with a T-cell phenotype (T-LAK cells) to EGFR positive bile duct carcinoma cells with dramatic enhancement of cytotoxicity in vitro. This specific killing of EGFR-positive cells was completely inhibited by parental mAb IgGs directed to EGFR and the CD3 antigen.Results When T-LAK cells were cultured with EGFR-positive tumor cells in the presence of Ex3 diabody, they produced much higher levels of IFN-, GM-CSF, and TNF- than in its absence, this being a possible mechanism underlying specific antitumor activity. The Ex3 diabody showed good stability when tested at 37°C for 48 h, and also markedly inhibited tumor growth of bile duct carcinoma xenografts in severe combined immunodeficient (SCID) mice. When Ex3 diabody (20 g/mouse) was administrated intravenously, together with T-LAK cells and interleukin-2 (IL-2), complete cure of tumors were observed in three of six mice, and the other three showed marked retardation of tumor growth.Conclusion The Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas.     

Protective role of cell division cycle 48 (CDC48) protein against neurodegeneration via ubiquitin-proteasome system dysfunction during zebrafish development

Cell division cycle 48 (CDC48), a ubiquitin-dependent molecular chaperone, is thought to mediate a variety of degradative and regulatory processes and maintain cellular homoeostasis. To investigate the protective function of CDC48 against accumulated ubiquitinated proteins during neurodevelopment, we developed an in vivo bioassay technique that detects expression and accumulation of fluorescent proteins with a polyubiquitination signal at the N terminus. When we introduced CDC48 antisense morpholino oligonucleotides into zebrafish embryos, the morphant embryos were lethal and showed defects in neuronal outgrowth and neurodegeneration, and polyubiquitinated fluorescent proteins accumulated in the inner plexiform and ganglion cell layers, as well as the diencephalon and mesencephalon, indicating that the degradation of polyubiquitinated proteins by the ubiquitin-proteasome system was blocked. These abnormal phenotypes in the morphant were rescued by CDC48 or human valosin-containing protein overexpression. Therefore, the protective function of CDC48 is essential for neurodevelopment.     

Up-regulation of pro-apoptotic protein Bim and down-regulation of anti-apoptotic protein Mcl-1 cooperatively mediate enhanced tumor cell death induced by the combination of ERK kinase (MEK) inhibitor and microtubule inhibitor

Blockade of the ERK signaling pathway by ERK kinase (MEK) inhibitors selectively enhances the induction of apoptosis by microtubule inhibitors in tumor cells in which this pathway is constitutively activated. We examined the mechanism by which such drug combinations induce enhanced cell death by applying time-lapse microscopy to track the fate of individual cells. MEK inhibitors did not affect the first mitosis after drug exposure, but most cells remained arrested in interphase without entering a second mitosis. Low concentrations of microtubule inhibitors induced prolonged mitotic arrest followed by exit of cells from mitosis without division, with most cells remaining viable. However, the combination of a MEK inhibitor and a microtubule inhibitor induced massive cell death during prolonged mitosis. Impairment of spindle assembly checkpoint function by RNAi-mediated depletion of Mad2 or BubR1 markedly suppressed such prolonged mitotic arrest and cell death. The cell death was accompanied by up-regulation of the pro-apoptotic protein Bim (to which MEK inhibitors contributed) and by down-regulation of the anti-apoptotic protein Mcl-1 (to which microtubule and MEK inhibitors contributed synergistically). Whereas RNAi-mediated knockdown of Bim suppressed cell death, stabilization of Mcl-1 by RNAi-mediated depletion of Mule slowed its onset. Depletion of Mcl-1 sensitized tumor cells to MEK inhibitor-induced cell death, an effect that was antagonized by knockdown of Bim. The combination of MEK and microtubule inhibitors thus targets Bim and Mcl-1 in a cooperative manner to induce massive cell death in tumor cells with aberrant ERK pathway activation.     

Nosocomial infection caused by vancomycin‐susceptible multidrug‐resistant Enterococcus faecalis over a long period in a university hospital in Japan

Compared with other developed countries, vancomycin‐resistant enterococci (VRE) are not widespread in clinical environments in Japan. There have been no VRE outbreaks and only a few VRE strains have sporadically been isolated in our university hospital in Gunma, Japan. To examine the drug susceptibility of Enterococcus faecalis and nosocomial infection caused by non‐VRE strains, a retrospective surveillance was conducted in our university hospital. Molecular epidemiological analyses were performed on 1711 E. faecalis clinical isolates collected in our hospital over a 6‐year period [1998–2003]. Of these isolates, 1241 (72.5%) were antibiotic resistant and 881 (51.5%) were resistant to two or more drugs. The incidence of multidrug resistant E. faecalis (MDR‐Ef) isolates in the intensive care unit increased after enlargement and restructuring of the hospital. The major group of MDR‐Ef strains consisted of 209 isolates (12.2%) resistant to the five drug combination tetracycline/erythromycin/kanamycin/streptomycin/gentamicin. Pulsed‐field gel electrophoresis analysis of the major MDR‐Ef isolates showed that nosocomial infections have been caused by MDR‐Ef over a long period (more than 3 years). Multilocus sequence typing showed that these strains were mainly grouped into ST16 (CC58) or ST64 (CC8). Mating experiments suggested that the drug resistances were encoded on two conjugative transposons (integrative conjugative elements), one encoded tetracycline‐resistance and the other erythromycin/kanamycin/streptomycin/gentamicin‐resistance. To our knowledge, this is the first report of nosocomial infection caused by vancomycin‐susceptible MDR‐Ef strains over a long period in Japan.     

Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)–variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH–VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.     

GEF-H1 Mediates Tumor Necrosis Factor-��-induced Rho Activation and Myosin Phosphorylation: ROLE IN THE REGULATION OF TUBULAR PARACELLULAR PERMEABILITY*

Tumor necrosis factor-α (TNF-α), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-α, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-α on the Rho pathway in tubular cells (LLC-PK₁ and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-α-induced alterations of paracellular permeability. We show that TNF-α induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-α-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-α-induced activation of these proteins. Importantly TNF-α enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-α-induced paracellular permeability increase was absent in LLC-PK₁ cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-α-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.Tumor necrosis factor-α (TNF-α)² is a pleiotropic proinflammatory cytokine that is synthesized as a membrane protein in response to inflammation, infection, and injury (1). Subsequently it is cleaved by the metalloprotease TNF-α convertase enzyme to release a 17-kDa soluble peptide (for a review, see Ref. 2). TNF-α has two receptors, the constitutively expressed, ubiquitous TNF receptor 1 and the inducible TNF receptor 2.An increasing body of evidence supports a key role for TNF-α in both acute renal injury and chronic kidney diseases (for reviews, see Refs. 3 and 4). Although TNF-α is almost undetectable in normal kidneys, elevated intrarenal, serum, or urine concentrations have been reported in various pathological states including ischemia-reperfusion, endotoxinemia, and early diabetic nephropathy (5–8). Moreover kidney injury in various pathological states was prevented or mitigated by inhibition of TNF-α production, by addition of neutralizing antibodies, or in TNF receptor knock-out mice (for a review, see Ref. 3). The central role of TNF-α in mediating kidney injury is therefore well established. Importantly TNF-α can be produced in the kidney not only by infiltrating macrophages and lymphocytes but by resident cells including the tubular epithelium. For example, in reperfusion injury TNF-α expression precedes macrophage infiltration and localizes mostly to the tubules (3, 7). Tubular TNF-α production is also enhanced by endotoxin and hypoxia (9–12). Although effects of locally released TNF-α on the tubular epithelium could contribute to its deleterious actions, the underlying mechanisms have been incompletely explored.Although a large number of studies have focused on the inflammatory and apoptotic signaling initiated by TNF-α in various cells, its cytoskeletal effects remain much less explored. In recent years Rho and its effector, Rho kinase (ROK), key regulators of both the actin cytoskeleton and myosin phosphorylation (13), have emerged as important mediators of TNF-α effects in endothelial cells (14–18). Similar effects in the tubular epithelium, however, have not been established. Even more importantly, the upstream signaling that connects the TNF receptor to activation of the Rho pathway remains completely unknown. Like other small GTPases, Rho cycles between an inactive (GDP-bound) and active (GTP-bound) form (13). The exchange of GDP to GTP during activation is stimulated by GDP-GTP exchange factors (GEFs). The diverse family of Rho GEFs contains >70 members in humans (19), making it challenging to identify the specific factors involved in mediating Rho activation through receptor-mediated stimuli. In the case of TNF-α, neither the particular Rho GEF involved nor the mechanism of its regulation has been identified in any of the cell systems studied.A rise in epithelial paracellular permeability through the intercellular junctions is a prominent event during inflammation (“leaky epithelium”) (for reviews, see Refs. 20 and 21). In addition, the junctions maintain the polarized phenotype of epithelial cells that is necessary for directional transport processes and constitute an important signaling platform that transmits environmental cues to the cells. Therefore, the consequences of junction disruption during inflammation might go beyond the compromised barrier functions. Interestingly TNF-α has been reported to affect the permeability of the tubular epithelium. Mullin et al. (22) have reported that in a tubular cell line TNF-α induced a temporary elevation in transepithelial resistance followed by a drop in transepithelial resistance and increased paracellular permeability. The transepithelial resistance decrease was blocked by genistein, a general tyrosine kinase inhibitor; however, the exact mechanism underlying the observed permeability changes remained incompletely explored.The actin cytoskeleton and especially phosphorylation of myosin light chain (MLC) was shown to be essential for the permeability increase caused by pathogens, cytokines, and growth factors in various epithelial and endothelial systems (for reviews, see Refs. 21, 23, and 24). Interestingly although myosin phosphorylation mediates the TNF-α-elicited permeability changes in intestinal cells (25, 26), phospho-MLC was reported not to be involved in the TNF-α-induced permeability rise in endothelial cells (17). The possible role of the Rho pathway and myosin phosphorylation in the TNF-α-induced permeability changes in the tubular epithelium therefore remains to be established.The aim of this study was to explore the signaling pathways through which TNF-α causes cytoskeleton remodeling and elevates paracellular permeability in kidney tubular cells. Our findings show that TNF-α induces rapid activation of RhoA that leads to Rho/Rho kinase-dependent actin remodeling and myosin phosphorylation. Using an affinity precipitation assay followed by mass spectrometry, we identified GEF-H1 as a TNF-α-activated GEF. We showed that GEF-H1 mediates the TNF-α-induced stimulation of Rho and its effectors. In addition, activation of the GEF-H1/Rho pathway by TNF-α was downstream of ERK signaling and required GEF-H1 phosphorylation on Thr-678. Finally using a dominant negative MLC mutant, we showed that myosin phosphorylation is essential for the TNF-α-induced elevation in paracellular permeability.     

Cancer prevention by carotenoids

Various natural carotenoids seem to be valuable for cancer prevention, and these carotenoids may be more suitable in combinational use, rather than in single use. In fact, we have proven that combinational use of natural carotenoids resulted in significant suppression of liver cancer. Patients of viral hepatitis with cirrhosis were administered with β-cryptoxanthin-enriched Mandarin orange juice, in addition to capsules of carotenoids mixture. Cumulative incidence of hepatocellular carcinoma development was compared with that in the group treated with carotenoids mixture capsules alone, or in the group without treatment (control group). In the data analysis at year 2.5, cumulative incidence of liver cancer in β-cryptoxanthin-enriched orange juice with carotenoids mixture capsules-treated group was lower than that in the control group (p = 0.05). Cumulative incidence of liver cancer in the group treated with carotenoids mixture capsules alone was also lower than that in the control group, but not statistically significant.     

Gene expression and localization of diacylglycerol kinase isozymes in the rat spinal cord and dorsal root ganglia

The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKζ and DGKι in the DRG. By immunohistochemical analysis, DGKζ immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKι immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKζ in the nucleus and DGKι in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKζ between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.The work was supported by grants-in-aid from the Ministry of Education, Science, Culture, and Sports of Japan (M.T., K.G.) and from the Ono Medical Research Foundation, Kato Memorial Bioscience Foundation, and Janssen Pharmaceutical (K.G.) and by the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Natural recovery of steppe vegetation on vehicle tracks in central Mongolia

Steppe desertification due to vehicle travel is a severe environmental issue in Mongolia. We studied natural vegetation recovery on abandoned vehicle tracks in the central Mongolia steppe through vegetation surveys and stable isotopic techniques. The following issues were addressed: (i) invasion of pioneering plant species, (ii) alteration of soil surface features, and (iii) contribution of revegetated plants to soil organic matter (SOM). The pioneering plant species that firstly invaded the abandoned tracks are those that could germinate, root and survive in the compacted track surface.Salsola collina is one of these candidate plants. Due to revegetation, soil surface hardness was reduced. With the improvement of surface microenvironmental conditions, other plants began to colonize and establish; concomitantly species richness and species diversity increased. Carbon isotope ratios of SOM at the top surface layer indicated that C₄-derived carbon contributed more to SOM in the early phase of recovery and decreased with further recovery     

Improving the accuracy of predicting secondary structure for aligned RNA sequences

Considerable attention has been focused on predicting the secondary structure for aligned RNA sequences since it is useful not only for improving the limiting accuracy of conventional secondary structure prediction but also for finding non-coding RNAs in genomic sequences. Although there exist many algorithms of predicting secondary structure for aligned RNA sequences, further improvement of the accuracy is still awaited. In this article, toward improving the accuracy, a theoretical classification of state-of-the-art algorithms of predicting secondary structure for aligned RNA sequences is presented. The classification is based on the viewpoint of maximum expected accuracy (MEA), which has been successfully applied in various problems in bioinformatics. The classification reveals several disadvantages of the current algorithms but we propose an improvement of a previously introduced algorithm (CentroidAlifold). Finally, computational experiments strongly support the theoretical classification and indicate that the improved CentroidAlifold substantially outperforms other algorithms.