Recombinant human fibroblast growth factor and autogenous bone for periodontal regeneration: Alone or in combination? A randomized clinical trial.

AIM: To compare the outcomes of therapy using recombinant human fibroblast growth factor (rhFGF)-2 combined with autologous bone grafting (ABG) therapy with those of rhFGF-2 alone and ABG alone in the treatment of periodontal intraosseous defects. METHODS: Periodontal intraosseous defects were randomized to receive rhFGF-2 therapy + ABG, rhFGF-2 therapy alone, or ABG alone. Periodontal examination and periapical radiography were performed preoperatively and at 3, 6, and 12 months postoperatively. RESULTS: At the 12 months follow-up, all three groups showed significant improvement in the clinical attachment level (CAL): 5.6 ± 1.6, 5.8 ± 1.7, and 5.2 ± 1.6 mm in the rhFGF-2 + ABG, rhFGF-2 alone, and ABG alone groups, respectively, with no significant inter-group differences (p < .05). rhFGF-2 therapy (alone or in combination) resulted in greater bone defect filling (BDF) (2.3 ± 1.2 mm and 2.6 ± 1.9 mm, respectively) than ABG therapy alone (1.2 ± 1.2 mm). Gingival recession was lesser in the ABG alone (1.2 ± 1.1 mm) and rhFGF-2 + ABG groups (1.4 ± 0.8 mm) than in the rhFGF-2 alone group (2.2 ± 1.2 mm). CONCLUSION: The results of this study showed that at 12 months postoperatively, all treatments resulted in statistically significant clinical improvements compared to the baseline. From these results, it can be concluded that rhFGF-2 promotes hard tissue regeneration in intraosseous defects.

Physical characterization of meso-erythritol as a crystalline bulking agent for freeze-dried formulations.

The purpose of this study was to identify and characterize new crystalline bulking agents applicable to freeze-dried pharmaceuticals. Thermal analysis of heat-melt sugar and sugar alcohol solids as well as their frozen aqueous solutions showed high crystallization propensity of meso-erythritol and D-mannitol. Experimental freeze-drying of the aqueous meso-erythritol solutions after their cooling by two different methods (shelf-ramp cooling and immersion of vials into liquid nitrogen) resulted in cylindrical crystalline solids that varied in appearance and microscopic structure. Powder X-ray diffraction and thermal analysis indicated different crystallization processes of meso-erythritol depending on the extent of cooling. Cooling of the frozen meso-erythritol solutions at temperatures lower than their Tg' (glass transition temperature of maximally freeze-concentrated phase, -59.7°C) induced a greater number of nuclei in the highly concentrated solute phase. Growth of multiple meso-erythritol anhydride crystals at around -40°C explains the powder-like fine surface texture of the solids dried after their immersion in liquid nitrogen. Contrarily, shelf-ramp cooling of the frozen solution down to -40°C induced an extensive growth of the solute crystal from a small number of nuclei, leading to scale-like patterns in the dried solids. An early transition of the freezing step into primary drying induced collapse of the non-crystalline region in the cakes. Appropriate process control should enable the use of meso-erythritol as an alternative crystalline bulking agent in freeze-dried formulations.

Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells.

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.

Viral envelope protein gp64 transgenic mouse facilitates the generation of monoclonal antibodies against exogenous membrane proteins displayed on baculovirus.

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.

A novel therapeutic approach for thrombocytopenia by minibody agonist of the thrombopoietin receptor.

Antibodies have brought valuable therapeutics in the clinical treatment of various diseases without serious adverse effects through their intrinsic features such as specific binding to the target antigen with high affinity, clinical safety as serum proteins, and long half-life. Agonist antibodies, furthermore, could be expected to maximize the value of therapeutic antibodies. Indeed, several IgG/IgM antibodies have been reported to induce cellular growth/differentiation and apoptosis. These agonist antibodies, however, should be further improved to exert more potent biologic activities and appropriate serum half-life depending upon the disease indications. Here, we report that IgG antibodies against the thrombopoietin receptor (Mpl), which have an absence or very weak agonist activity, can be engineered to be agonist minibodies, which include diabody or sc(Fv)2 as potent as natural ligand. Through this technological development, minibodies have been successfully constructed to bind and activate 2 types of dysfunctional mutant Mpls that cause congenital amegakaryocytic thrombocytopenia (CAMT). This drastic conversion of biologic activities by designing minibodies can be widely applicable to generate agonist minibodies for clinical application, which will constitute a new paradigm in antibody-based therapeutics.

Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma.

For detection of hepatocellular carcinoma (HCC) in patients with liver cirrhosis, serum alpha-fetoprotein has been widely used, but its sensitivity has not been satisfactory, especially in small, well-differentiated HCC, and complementary serum marker has been clinically required. Glypican-3 (GPC3), a heparan sulfate proteoglycan anchored to the plasma membrane, is a good candidate marker of HCC because it is an oncofetal protein overexpressed in HCC at both the mRNA and protein levels. In this study, we demonstrated that its NH(2)-terminal portion [soluble GPC3 (sGPC3)] is cleaved between Arg(358) and Ser(359) of GPC3 and that sGPC3 can be specifically detected in the sera of patients with HCC. Serum levels of sGPC3 were 4.84 +/- 8.91 ng/ml in HCC, significantly higher than the levels seen in liver cirrhosis (1.09 +/- 0.74 ng/ml; P < 0.01) and healthy controls (0.65 +/- 0.32 ng/ml; P < 0.001). In well- or moderately-differentiated HCC, sGPC3 was superior to alpha-fetoprotein in sensitivity, and a combination measurement of both markers improved overall sensitivity from 50% to 72%. These results indicate that sGPC3 is a novel serological marker essential for the early detection of HCC.

Efficient 340-nm light generation by a ridge-type waveguide in a first-order periodically poled MgO:LiNbO3.

Quasi-phase-matched (QPM) UV second-harmonic generation (SHG) in a periodically poled MgO:LiNbO3 waveguide is presented. A ridge-type waveguide with high nonlinearity and strong resistance to photorefractive damage was achieved by use of an ultraprecision machining technique. By use of this waveguide in 1.4-microm periodically poled MgO:LiNbO3, a first-order QPM SHG device for 340-nm UV radiation was demonstrated. In a single-pass configuration, continuous-wave 22.4-mW UV light was generated for a fundamental power of 81 mW, corresponding to a normalized conversion efficiency of 340%/W.