Analysis of effects and pharmacokinetics of subcutaneously administered BDNF.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and has been shown to be a potent and effective trophic factor for motor neurons and other neurons of the peripheral and central nervous. Little is known, however, about the relationship between the efficacy and pharmacokinetics of s.c. administered BDNF. In this study, the efficacy of BDNF on motor neuron protection in sciatic or facial nerve axotomy models was examined and compared with the concommitant concentrations of BDNF in plasma. Delayed treatment (started at 1 week after surgery) of BDNF was also shown to retard choline acetyltransferase reduction in sciatic nerve axotomy models.

Antagonistic effect of synthetic peptides corresponding to the binding regions within fimbrial subunit protein from Porphyromonas gingivalis to human gingival fibroblasts.

Specific binding region within fimbrial subunit protein (fimbrilin) from Porphyromonas gingivalis strain 381 was studied in cultured human gingival fibroblasts. Fluorescent micrographs visualised FITC-labelled fimbriae of P. gingivalis specifically bound to normal human fibroblast cell line (Gin-1) along the cell surface. Flow cytometric analysis also revealed the binding of FITC-labelled fimbriae to Gin-1 cells. Synthetic peptides composed of residues 1-20 (AFGVGDDESKVAKLTVMVYN) of the fimbrilin from P. gingivalis, FP381 (1-20), FP381 (69-80; ALTTELTAENQE) and FP381 (171-181; DA-NYLTGSLTT) definitely inhibited P. gingivalis fimbria-binding to Gin-1 cells by enzyme-linked immunosorbent assay (ELISA). Furthermore, based on the Scatchard plot analysis of the binding of 125I-labelled P. gingivalis fimbriae to Gin-1 cells, the apparent dissociation constant (Kd) was calculated as 15.9 pM, and the number of binding sites (Rt) was estimated as 150 sites/cell. Binding studies of 125I-labelled FP381(171-181) also revealed the presence of a non-interacting, single class of affinity binding sites: the apparent Kd and Rt were 29.2 nM and 18440 sites/cell on Gin-1 cells, respectively. These results demonstrate that specific binding regions on P. gingivalis fimbriae to human gingival fibroblasts are present, and certain corresponding peptides clearly inhibited the binding of P. gingivalis fimbriae to human gingival fibroblasts.

Human astrocytoma cells (U-87 MG) exhibit a specific substance P binding site with the characteristics of an NK-1 receptor.

To investigate substance P (SP) receptors on an established human astrocytoma cell line (U-87 MG), [3H][Sar9,Met(O2)11]-SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti-SP binding protein antibody. In U-87 MG and U-373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U-87 MG cell membrane-enriched preparations, the binding of [3H][Sar9,Met(O2)11]-SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent KD of 1.15 +/- 0.15 nM and a Bmax of 108 +/- 9.8 fmol/mg of protein. [3H][Sar9, Met(O2)11]-SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl-5'-imidodiphosphate, but not GDP and GMP, reduced the Bmax without changing the affinity of [3H][Sar9,Met(O2)11]-SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens culinaris agglutinin (LCA)] to determine the nature of carbohydrate chains on the U-87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U-87 MG have either a biantennary complex-type or a high mannose-type of carbohydrate chain and may be regulated by GTP-binding protein(s).

Chemotaxis of human monocytes by synthetic peptides that mimic segments of Porphyromonas gingivalis fimbrial protein.

Porphyromonas gingivalis strain 381 fimbriae and their synthetic peptide segments were assessed for migration-stimulating activity on human peripheral blood monocytes by multiwell chemotaxis assay. P. gingivalis 381 fimbrial protein was found to markedly enhance migration of human monocytes. The observed increase in monocyte migration occurred mainly directed toward a positive stimulus (chemotaxis). Furthermore, lipopolysaccharides extracted from P. gingivalis 381 were shown to induce chemotaxis and chemokinesis. It was also revealed that the migration of monocytes was increased by specific synthetic peptide segments, FP381(61-80) and FP381(171-185), that correspond to GKTLAEVKALTTELTAENQE and DANYLTGSLTTFNGA, respectively, based on the amino acid sequence of the fimbrial subunit protein proposed by Dickinson et al., and the migration stimulation was ascribed to chemotaxis. Furthermore, within the amino acid sequences, the LTXXLTXXN sequence may play an important role in binding the organisms to monocytes and macrophages and in the induction of migration-stimulating activity.

Modulation of substance P/neurokinin-1 receptor in human astrocytoma cells by antisense oligodeoxynucleotides.

1. An antisense oligodeoxynucleotide corresponding to the NH2-terminus of the substance P (SP)/neurokinin-1 (SP/NK1) receptor was constructed and added to cultures of human astrocytoma U-87 MG cells in vitro and rats in vivo. 2. Antisense oligodeoxynucleotide at a concentration of 30 microM progressively reduced the specific binding [3H][Sar9, Met(O2)11]-SP, selective SP/NK1 receptor agonist, in the U-87 MG astrocytoma cells by approx. 31% on the second day after treatment (control: 26.1 +/- 2.4 fmol/mg protein vs antisense oligodeoxynucleotide: 18.0 +/- 1.4 fmol/mg protein, P < 0.001). 3. Treatment with 30 microM antisense oligodeoxynucleotide for 2 days inhibited the SP/MK1 receptor-induced influx of 45Ca2+ into the U-87 MG cells by approx. 35%. 4. When the same antisense oligodeoxynucleotides were encapsulated in liposomes and injected into the lateral cerebral ventricle of rats, functional SP receptor was blocked.

Humoral and cellular immune responses to the fimbriae of Porphyromonas gingivalis and their synthetic peptides.

Subcutaneous injection of fimbriae from Porphyromonas gingivalis strain 381 in Freund's incomplete adjuvant (FIA) resulted in an excellent serum anti-fimbrial immunoglobulin G (IgG) response in guinea-pigs and BALB/c mice. Administration of P. gingivalis fimbriae also elicited distinct cellular immune responses to the fimbriae in terms of ear lobe reaction in BALB/c but not in BALB/c nu/nu mice, and of skin reaction in guinea-pigs. When the guinea-pigs were given a semi-synthetic adjuvant GM-53--sodium beta-N-acetylglycosaminyl-(1-->4)-N-acetylmuramyl-L-alanyl-D-isogl utaminyl- (L)-stearoyl-(D)-meso-2, 6-diaminopimelic acid-(D)-amide-D-alanine--and fimbriae in FIA by subcutaneous injection, more enhanced production of serum anti-fimbrial IgG and stronger cellular immune responses were induced in the guinea-pigs than in those given fimbriae alone. Synthetic peptide FP381(202-221), which corresponds to the amino-acid residue numbers 202-221 based on the amino-acid sequence of fimbrilin from P. gingivalis strain 381, elicited humoral and cellular immune responses in guinea-pigs immunised with the fimbriae or FP381(202-221). Furthermore, subcutaneous administration of synthetic peptide FP381(61-80) with GM-53 induced lesser degrees of humoral and cellular immune responses in guinea-pigs than did FP381(202-221). However, when the fimbriae or FP381(61-80) were administered with bovine serum albumin (BSA), markedly elevated levels of specific anti-BSA antibody were seen in the serum of BALB/c mice. These results clearly indicated that fimbriae from P. gingivalis 381 and their oligopeptide segments induced humoral and cellular immune responses and exhibited immuno-adjuvant activities in guinea-pigs and BALB/c mice.

Immunobiological activities of synthetic peptide segments of fimbrial protein from Porphyromonas gingivalis.

Several oligopeptide segments of fimbrial subunit protein (fimbrilin) of Porphyromonas gingivalis strain 381 were synthesized and tested for immunobiological activities. Peptides F3(31-50; amino acid residue numbers 31 to 50, based on the amino acid sequence of the fimbrilin proposed by Dickinson et al., Infect. Immun., 170, 1658, 1988), F12(212-231) and F17(312-331) were found to be immunodominant epitopes of this fimbrial protein as revealed by ELISA. Furthermore, peptides F5(71-90) and F17(312-331) were demonstrated to agglutinate rabbit erythrocytes, and were mitogenic for BALB/c spleen cells but not thymocytes. These peptides enhanced the number of fimbria-specific antibody-secreting cells in BALB/c spleen cell cultures, and induced cytokines such as tumor necrosis factor-alpha and interleukin-6 production in human monocyte/macrophage cultures. The data demonstrate that these defined peptide segments are responsible for the immunostimulating portions within the fimbrial protein molecule.