T-448, a specific inhibitor of LSD1 enzyme activity, improves learning function without causing thrombocytopenia in mice.

Dysregulation of histone H3 lysine 4 (H3K4) methylation has been implicated in the pathogenesis of several neurodevelopmental disorders. Targeting lysine-specific demethylase 1 (LSD1), an H3K4 demethylase, is therefore a promising approach to treat these disorders. However, LSD1 forms complexes with cofactors including growth factor independent 1B (GFI1B), a critical regulator of hematopoietic differentiation. Known tranylcypromine-based irreversible LSD1 inhibitors bind to coenzyme flavin adenine dinucleotide (FAD) and disrupt the LSD1-GFI1B complex, which is associated with hematotoxicity such as thrombocytopenia, representing a major hurdle in the development of LSD1 inhibitors as therapeutic agents. To discover LSD1 inhibitors with potent epigenetic modulation and lower risk of hematotoxicity, we screened small molecules that enhance H3K4 methylation by the inhibition of LSD1 enzyme activity in primary cultured rat neurons but have little impact on LSD1-GFI1B complex in human TF-1a erythroblasts. Here we report the discovery of a specific inhibitor of LSD1 enzyme activity, T-448 (3-((1S,2R)-2-(cyclobutylamino)cyclopropyl)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzamide fumarate). T-448 has minimal impact on the LSD1-GFI1B complex and a superior hematological safety profile in mice via the generation of a compact formyl-FAD adduct. T-448 increased brain H3K4 methylation and partially restored learning function in mice with NMDA receptor hypofunction. T-448-type LSD1 inhibitors with improved safety profiles may provide unique therapeutic approaches for central nervous system disorders associated with epigenetic dysregulation.

Identification of the cancer cell proliferation and survival functions of proHB-EGF by using an anti-HB-EGF antibody.

PURPOSE: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The membrane-bound proHB-EGF is known to be a precursor of the soluble form of HB-EGF (sHB-EGF), which promotes cell proliferation and survival. While the functions of sHB-EGF have been extensively studied, it is not yet fully understood if proHB-EGF is also involved in cellular signaling events. In this study, we utilized the anti-HB-EGF monoclonal antibodies Y-142 and Y-073, which have differential specificities toward proHB-EGF, in order to elucidate proHB-EGF functions in cancer cells. EXPERIMENTAL DESIGN: The biological activities of proHB-EGF were assessed in cell proliferation, caspase activation, and juxtacrine activity assays by using a 3D spheroid culture of NUGC-3 cells. RESULTS: Y-142 and Y-073 exhibited similar binding and neutralizing activities for sHB-EGF. However, only Y-142 bound to proHB-EGF. We could detect the function of endogenously expressed proHB-EGF in a 3D spheroid culture. Blocking proHB-EGF with Y-142 reduced spheroid formation, suppressed cell proliferation, and increased caspase activation in the 3D spheroid culture of NUGC-3 cells. CONCLUSIONS: Our results show that proHB-EGF acts as a cell proliferation and cell survival factor in cancer cells. The results suggest that proHB-EGF may play an important role in tumor progression.

Characterization of a variety of neutralizing anti-heparin-binding epidermal growth factor-like growth factor monoclonal antibodies by different immunization methods.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The accumulated evidence on the tumor-progressing roles of HB-EGF has suggested that HB-EGF-targeted cancer therapy is expected to be promising. However, the generation of neutralizing anti-HB-EGF monoclonal antibodies (mAbs) has proved difficult. To overcome this difficulty, we performed a hybridoma approach using mice from different genetic backgrounds, as well as different types of HB-EGF immunogens. To increase the number of hybridoma clones to screen, we used an electrofusion system to generate hybridomas and a fluorometric microvolume assay technology to screen anti-HB-EGF mAbs. We succeeded in obtaining neutralizing anti-HB-EGF mAbs, primarily from BALB/c and CD1 mice, and these were classified into 7 epitope bins based on their competitive binding to the soluble form of HB-EGF (sHB-EGF). The mAbs showed several epitope bin-dependent characteristics, including neutralizing and binding activity to human sHB-EGF, cross-reactivity to mouse/rat sHB-EGF and binding activity to the precursor form of HB-EGF. The neutralizing activity was also validated in colony formation assays. Interestingly, we found that the populations of mAb bins and the production rates of the neutralizing mAbs were strikingly different by mouse strain and by immunogen type. We succeeded in generating a variety of neutralizing anti-HB-EGF mAbs, including potent sHB-EGF neutralizers that may have potential as therapeutic agents for treating HB-EGF-dependent cancers. Our results also suggest that immunization approaches using different mouse strains and immunogen types affect the biological activity of individual neutralizing antibodies.

In-vitro and in-vivo study of periodontal ligament cryopreserved with a magnetic field.

INTRODUCTION: The purpose of this study was to examine the effect of a new cryopreservation method with a magnetic field on periodontal regeneration in vitro and in vivo. METHODS: Human periodontal ligament cells were frozen in 10% dimethyl sulfoxide by using a programmed freezer with a magnetic field. Cells were cryopreserved for 3 days at -150°C. Immediately after thawing, collagen type I and alkaline phosphatase gene expression were determined by real-time polymerase chain reaction. Incisors were extracted from 15-week-old Wistar rats and cryopreserved or dried for 3 days. Then the incisors were replanted into the same sockets. Ninety days after transplantation, they were observed under light microscopy. RESULTS: There was no difference in the messenger RNA expression of collagen type I between the cryopreserved and the control groups. The expression of alkaline phosphatase messenger RNA in the cryopreserved group was slightly decreased compared with the control group. There was no progressive root resorption in the teeth that were replanted immediately (control group) or cryopreserved. However, there was widespread root resorption and ankylosis in the dried teeth. CONCLUSIONS: These results show that a magnetic field programmed freezer can be successfully used for cryopreservation of teeth.

Influences of reduced masticatory sensory input from soft-diet feeding upon spatial memory/learning ability in mice.

It has been reported that reduction of masticatory afferent stimulation might influence learning and memory function. In order to clarify the influences of reduced masticatory sensory input on spatial memory/learning ability and neuropathological changes, we conducted the Morris water maze experiment and investigated the number of hippocampal neurons in association with the differences in masticatory afferent stimuli from hard- and soft-diet feeding in mice. The water maze experiment showed no significant difference in learning ability between 180-day-old solid- and powderdiet groups. Meanwhile, the ability was significantly reduced in the 360-day-old powder-diet group as compared with the age-matched solid-diet group. The total number of pyramidal cells in the hippocampal CA1 and CA3 regions was significantly smaller in 360-day-old powder-diet group than in the remaining groups. These results demonstrate that reduction of masticatory afferent stimuli due to long-term soft-diet feeding may induce neuron loss in the hippocampus and reduced memory/learning ability.

Neutralizing effects of an anti-vascular endothelial growth factor antibody on tooth movement.

Our recent studies demonstrated that local administration of recombinant human vascular endothelial growth factor (rhVEGF) during experimental tooth movement enhanced the number of osteoclasts and the rate of tooth movement. The purpose of this study was to examine the effect of anti-VEGF polyclonal antibody on osteoclastic differentiation, the amount of tooth movement, and the degree of tooth relapse in 30-day-old mice. First, these mice were subjected to various doses of anti-VEGF polyclonal antibody, with tooth movement for three days. In the next study, daily injections of 10-microg antibody were administered for 18 days during the experimental tooth movement. The amount of tooth movement was measured as in our previous study. Furthermore, in the third study, we administered daily injection of 10-microg antibody and measured tooth relapse after the experimental tooth movement for 45 days. The osteoclasts number in 10- and 50-microg antibody two-time injection group was significantly smaller than that in the controls (P < .05). The number of osteoclasts was decreased more substantially by daily injection of 10-microg antibody, showing more significant differences from the controls (P < .01). The amount of tooth movement was significantly less in the experimental group than in the controls on days 15 and 18 (P < .05). Furthermore, the amount of relapse in the experimental group was significantly less than that in the controls on days 9 and 11 after removal of the appliance (P < .05). These results show that the treatment of anti-VEGF polyclonal antibody markedly reduced the osteoclasts number and inhibited the amount of tooth movement and relapse of moved teeth.

Guided bone regeneration to repair an alveolar bone defect in a girl whose cleft lip and palate had been repaired.

We report the case of a girl aged 10 years whose alveolar bony defect was closed with a polytetrafluoroethylene membrane, which was incorporated in bone after 4 months. We suggest that the principle of guided bone regeneration can be used to repair cleft defects provided that the mucoperiosteal flaps are handled carefully and that good anti-infective measures are taken to prevent early exposure and microbial contamination of the membrane barrier.