The role of Nod1 signaling in corneal neovascularization.

PURPOSE: Corneal neovascularization (CNV) is associated with Chlamydia trachomatis. The minimal components of bacterial cell walls are recognized by nucleotide-binding oligomerization domain-containing protein (Nod), which is important for host defense--a mechanism manifested in human corneal cells. We aimed to examine whether Nod stimulation is associated with CNV. METHODS: Three groups of mice with alkali-induced CNV were topically treated with tripeptide L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP, a Nod1 agonist), muramyl dipeptide (a Nod2 agonist), or phosphate-buffered saline twice daily for 8 days. The time course responses were quantified using biomicroscopic examinations and immunohistochemistry. Angiogenic factor expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction. To confirm the involvement of Nod1 signaling in CNV, RICK (an essential molecule in Nod signaling)-knockout mice treated with Tri-DAP were examined biomicroscopically and immunohistochemically 8 days after injury. RESULTS: According to the biomicroscopic camera images and histology, Tri-DAP and muramyl dipeptide promoted CNV. Significantly, Tri-DAP increased the number and size of the neovascularized areas. The messenger RNA expression level of vascular endothelial growth factor was elevated in the Tri-DAP-treated mice after alkali injury. Compared with wild-type mice, CNV was attenuated in RICK-deficient mice treated with Tri-DAP. CONCLUSIONS: These data suggest that Nod1 stimulation is an important inducer of CNV and that Nod1 might be useful in the development of CNV therapies.

Analgesic effects of chemically synthesized NOD1 and NOD2 agonists in mice.

Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors, NOD1 and NOD2, recognize the diaminopimelic acid (DAP)-containing peptide moiety and muramyldipeptide (MDP) moiety of bacterial peptidoglycan, respectively. Muramyldipeptide has been reported to exert analgesic activity to decrease the frequency of acetic acid-induced writhing movements in mice. In this study, we demonstrated the analgesic activities of NOD1 as well as NOD2 agonists. Intravenous injection of NOD2-agonistic MDP, 6-O-stearoyl-MDP (L18-MDP), and MDP-Lys (L18) exhibited analgesic activity at 10, 50, and 2.0 µg/head, respectively, in BALB/c mice. NOD1-Agonistic FK156 (D-lactyl-L-Ala-D-Glu-meso-DAP-L-Gly) and FK565 (heptanoyl-D-Glu-meso-DAP-D-Ala) were also analgesic at 50 µg/head and 1.0 µg/head, respectively. The analgesic effect of FK565 appeared from 30 min, reached maximum activity at 8 h, and continued until 24 h. The FK565 exhibited activity by various administration routes; intravenous, intraperitoneal, intramuscular, sublingual (1.0 µg/head each), subcutaneous, intragastric (oral), intragingival (10 µg/ head each) and intracerebroventricular (0.01 µg/head). The analgesic activity of FK565 was observed even in tumor necrosis factor (TNF)-α knockout, interleukin (IL)-1α/ß double knockout, and their triple knockout mice. Naloxane, a non-selective antagonist for the opioid receptor, completely inhibited the analgesic effect of FK565. These findings suggest that NOD1 and NOD2 activation induces an analgesic effect via opioid receptors in a TNF-α and IL-1α/ß independent manner.

Activation of tumoricidal properties in macrophages and inhibition of experimentally-induced murine metastases by a new synthetic acyltripeptide, FK-565.

The effect of FK-565, a novel low molecular weight (MW) acyltripeptide, on tumoricidal properties of murine macrophages is reported here. Peritoneal macrophages (PMs) harvested from C57BL/6 mice and beige mice were rendered cytotoxic to syngeneic B16 melanoma cells following their interaction in vitro with FK-565. Maximal and reproducible activation of tumoricidal properties in PM were obtained by interaction in vitro with 25 micrograms/ml of FK-565 for a 24 h period, and as little as 0.5 microgram/ml of FK-565 was sufficient to induce significant cytotoxicity. Murine PMs activated by FK-565 in vitro were cytotoxic to syngeneic and xenogeneic tumor cells, but did not affect allogeneic nontumor cells. The PMs were also activated to kill B16 melanoma cells by intraperitoneal injections of FK-565 (10 mg/kg). Multiple injections of FK-565 into mice also slightly but significantly inhibited lung metastases. These results suggest that FK-565 has potential as an effective immunopotentiator in immunotherapy.

Immunoactive peptides, FK-156 and FK-565. I. Enhancement of host resistance to microbial infection in mice.

The protective effect of an immunoactive peptide, D-lactoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(L)-glycine (FK-156) and a related compound, heptanoyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alanine (FK-565) was determined in mice with various kinds of microbial infections. FK-156 and FK-565 were given to mice either subcutaneously or orally before challenge. The drugs enhanced significantly the defense of mice against acute systemic infections induced by various extracellular and facultative intracellular organisms, and subcutaneous abscess by Staphylococcus aureus. The protective effect of these drugs against Escherichia coli infection differed considerably depending on the route of administration; FK-156 was only effective by the parenteral route; however, FK-565 was effective by both parenteral and oral routes. After subcutaneous dosing with FK-156, the enhancement of host defense of mice against E. coli infection was more rapid than against Listeria infection. The enhancing effects of FK-156 and FK-565 on host defense of mice against pseudomonal infection was more potent than other immunoactive drugs.

Immunoactive peptides, FK-156 and FK-565. II. Restoration of host resistance to microbial infection in immunosuppressed mice.

The immunoactive peptides, FK-156 and its analogue, FK-565 were evaluated in various models of mice immunosuppressed with cyclophosphamide, hydrocortisone, mitomycin C, carrageenan and tumor cells. Treatment with FK-156 (subcutaneous) and FK-565 (oral) markedly restored host defense ability against microbial infection. The therapeutic effect of ticarcillin or gentamicin alone against pseudomonal infection in cyclophosphamide- and hydrocortisone-treated mice and tumor-bearing mice was much lower than in normal mice. The therapeutic effect of these antibiotics against pseudomonal infection in immunosuppressed mice was enhanced markedly by combined use with FK-156. The killing ability of macrophages and polymorphonuclear leukocytes of the immunosuppressed mice was also markedly enhanced by dosing with FK-156.

Immunoactive peptides, FK-156 and FK-565. III. Enhancement of host defense mechanisms against infection.

We investigated the effect of immunoactive peptides, FK-156 and FK-565 on host defense mechanisms against microbial invasion. It was shown that these drugs given to normal mice increased the counts of phagocytes in both peripheral blood and peritoneal cavity, and enhanced the chemotactic, phagocytic and killing activities of peritoneal macrophages and polymorphonuclear leukocytes, and stimulated the phagocytic function of the reticuloendothelial system. Enhanced host resistance to microbial infection by these immunoactive peptides might be induced by both increase in counts and enhancement of functions of phagocytes. FK-156 restored decreased counts and functions of phagocytes in mice immunosuppressed by cyclophosphamide, hydrocortisone or tumor. These findings suggest that these immunoactive peptides could be applied to prevent intractable infection in immunocompromised hosts.

Antitumor effects of novel immunoactive peptides, FK-156 and its synthetic derivatives.

Effects produced by intratumor or systemic application of FK-156 and its synthetic derivatives on the syngeneic P388-DBA/2 mouse system were investigated. Among 21 compounds tested, FK-156, FK-565, FR-46758, FR-48217, FR-46091 and FR-47920 substantially suppressed tumor growth when directly injected into a tumor mass and further experiments showed that FK-156, FK-565 and FR-46758 were effective even when administered subcutaneously into site remote from tumor. The mechanisms of growth inhibition are strongly suggested to be host mediated, because these three compounds have remarkably low cytotoxicity against P388 cells in vitro. A single dose of FK-565, however, markedly decreased body weight in healthy DBA/2 mice, whereas FK-156 and FR-46758 did not. These results indicate the superiority of FK-156 and FR-46758 as immunotherapeutic agents over FK-565 with respect to their safety for treatment of cancer. Although significant life-span prolongation could not be seen in the two-injection regimen of six compounds in either system, systemic multiple injections of FK-156 and FR-46758 provided a statistically significant increase in the median survival time of P388 tumor bearing mice.