Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.     

Determination of Pseudomonas aeruginosa by Biochemical Test Methods

A rapid and simple test method for the detection of acylamidase activity of Pseudomonas aeruginosa was devised. One loopful of a nutrient agar overnight culture of a test organism was inoculated into 1 ml of a test medium consisting of 0.2% KH₂PO₄, 0.01% MgSO₄·7H₂O, 0.5% NaCl and 0.1% acetamide (final pH 6.8). After aerobic incubation at 37C for 6 hr, one drop of Nessler's reagent was dropped into the test medium. A reddish-brown sediment appeared immediately if results were positive. Of 40 test strains of P. aeruginosa 39 gave strongly positive results. A strain showed a weakly positive result after 6 hr incubation, but the reaction became stronger after 18 hr culture. Other species of Pseudomonas, and various species of bacteria such as genera Vibrio and Aeromonas, and family Enterobacteriaceae were negative in this test. From these experimental results, the acylamidase test was considered to be highly specific for strains of P. aeruginosa, and therefore useful as a reliable method for the identification of this species.     

Rapid suppression of protein degradation in skeletal muscle after oral feeding of leucine in rats

A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation in rats and mice. This study determined whether dietary amino acids inhibit postprandial protein degradation in rat skeletal muscle. When rats fed on a 20% casein diet for 1 h after 18 h starvation, the rate of myofibrillar protein degradation measured by N(tau)-methylhistidine release from the isolated extensor digitorum longus muscle was significantly (p < 0.05) decreased at 4 h after refeeding. A diet containing an amino acid mixture which is the same composition as casein also reduced myofibrillar protein degradation at 4 h after refeeding (p < 0.05). An essential amino acid mixture (15.1%, corresponding to casein composition) and a leucine (2.9%) diets reduced the rate of myofibrillar protein degradation after refeeding (p < 0.05), whereas a protein free diet did not. Administration of leucine alone (0.135 g/100 g body weight) by a feeding tube induced a decrease in the rate of myofibrillar protein degradation at 2 h after administration (p < 0.05), whereas the serum insulin concentration was constant after leucine administration. These results suggested that leucine is one of regulating factors of myofibrillar protein degradation after refeeding of a protein diet.     

Effect of continuous negative-pressure breathing on skin blood flow during exercise in a hot environment

To assessthe impact of continuous negative-pressure breathing (CNPB) on theregulation of skin blood flow, we measured forearm blood flow (FBF) byvenous-occlusion plethysmography and laser-Doppler flow (LDF) at theanterior chest during exercise in a hot environment (ambienttemperature = 30°C, relative humidity = ~30%). Seven malesubjects exercised in the upright position at an intensity of 60% peakoxygen consumption rate for 40 min with and without CNPB after 20 minof exercise. The esophageal temperature(T_es) in both conditionsincreased to 38.1°C by the end of exercise, without any significantdifferences between the two trials. Mean arterial pressure (MAP)increased by ~15 mmHg by 8 min of exercise, without any significantdifference between the two trials before CNPB. However, CNPB reducedMAP by ~10 mmHg after 24 min of exercise (P < 0.05). The increasein FBF and LDF in the control condition leveled off after 18 min ofexercise above a T_es of37.7°C, whereas in the CNPB trial the increase continued, with arise in T_es despite the decreasein MAP. These results suggest that CNPB enhances vasodilation of skinabove a T_es of ~38°C bystretching intrathoracic baroreceptors such as cardiopulmonarybaroreceptors.

     

Two Novel Endopeptidases Released into the Medium during Mating of Gametes of Chlamydomonas reinhardtii

During the mating reaction between mt⁺ and mt^- gametes of Chlamydomonasreinhardtii, two novel endopeptidases, each of which was ableto digest the B chain of insulin, were released into the culturemedium, together with a gamete lytic enzyme (GLE) which is responsiblefor digestion of the gametic cell walls. Both endopeptidasesand GLE were copurified from the mating medium by column chromatographyon DEAE-cellulose and concanavalin A. Gel filtration separatedthe peptidases, which were unable to digest gametic cell walls,into two fractions, endopeptidase-1 and endopeptidase-2. Theseenzymes were also separated from GLE, which was unable to digestthe B chain of insulin. Endopeptidase-1, with a molecular massof about 200 kDa, cleaved the B chain of insulin at the Ala¹⁴-Leu¹⁵peptide bond, and this activity was inhibited by EDTA. Endopeptidase-2,with a molecular mass of about 110 kDa, digested the peptideat the Leu¹⁵-Tyr¹⁶ peptide bond and was sensitive to iodoacetateand chymostatin. When the cell walls of gametes of either mating-typewere digested prior to mating with exogenously added GLE, thetwo endopeptidases were released into the medium, a result thatsuggests that they are stored, like GLE, outside the plasmalemma. (Received March 25, 1994; Accepted June 13, 1994)     

In vivo effects of recombinant human lymphotoxin on human medulloblastoma xenograft: Enhancement of antitumor activity of etoposide

The authors investigated the antitumor activities of rHuLT alone and in combination with etoposide on human medulloblastoma xenografts growing subcutaneously in nude mice. Intravenous administration of rHuLT (1.0×10⁵U/kg, 5.0×10⁵U/kg, 2.5×10⁶U/kg, three times a week for three weeks) suppressed medulloblastoma growth depending on the dose. However, the highest dosage caused serious side effects. Combining rHuLT (intravenously, 5.0×10⁵U/kg, three times a week for three weeks) with etoposide (intraperitoneally, 20mg/kg, once a week for three weeks) increased the antitumor activity without causing serious toxicity. Microscopically, tumor specimen showed thrombosed tumor vessels and massive necrosis 3 weeks after rHuLT treatment. Ultrastructural examination revealed that 120 minutes after the administration of rHuLT alone, disruption of interendothelial junctions was evident, and that the endothelial cells were destroyed at 240 minutes.Concentration of etoposide in tumor tissue peaked 30 minutes after intraperitoneal administration, and then decreased with time. When etoposide was administered in combination with rHuLT, the concentration of etoposide in tumor tissue after 60 to 240 minutes was significantly higher than when etoposide was given alone, and the area under the concentrationversus time curve was also greater for the tumors of mice with combination treatment.The findings suggest that the proper combination of rHuLT and etoposide may have synergistic antitumor activities. Histological changes suggest that increased concentrations of etoposide within the tumor after combination therapy may occur due to increased vascular permeability and/or decreased etoposide clearance which is the result of blood stasis in the tumor vasculature.Abbreviations AUC area under the concentration versus time curve - LT lymphotoxin - rHuLT recombinant human lymphotoxin - rHuTNF recombinant human tumor necrosis factor - TNF tumor necrosis factor     

Design, synthesis and structure-affinity relationships of aryloxyanilide derivatives as novel peripheral benzodiazepine receptor ligands

Since the peripheral benzodiazepine receptor (PBR) has been primarily found as a high-affinity binding site for diazepam in rat kidney, numerous studies of it have been performed. However, the physiological role and functions of PBR have not been fully elucidated. Currently, we presented the pharmacological profile of two high and selective PBR ligands, N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl)acetamide (7-096, DAA1106) (PBR: IC(50)=0.28 nM) and N-(4-chloro-2-phenoxyphenyl)-N-(2-isopropoxybenzyl)acetamide (7-099, DAA1097) (PBR: IC(50)=0.92 nM). The compounds are aryloxyanilide derivatives, and identified with known PBR ligands such as benzodiazepine (1, Ro5-4864), isoquinoline (2, PK11195), imidazopyridine (3, Alpidem), and indole (5, FGIN-1-27) derivatives. The aryloxyanilide derivatives, which have been derived by opening the diazepine ring of 1, are a novel class as PBR ligands and have exhibited high and selective affinity for peripheral benzodiazepine receptors (PBRs). These novel derivatives would be useful for exploring the functions of PBR. In this paper, the design, synthesis and structure-affinity relationships of aryloxyanilide derivatives are described.     

Liposomal lactoferrin induced significant increase of the interferon-alpha (IFN-alpha) producibility in healthy volunteers

Interferon-alpha (IFN-alpha) producibility has been widely accepted as one of the important markers to evaluate the immune status. In this study, preliminary clinical tests were carried out to confirm the immunomodulatory activity of liposomal lactoferrin including IFN-alpha producibility and NK activity. In a primary open trial, the liposomal lactoferrin was administered to five healthy males for one week and various immunological indices were evaluated. Furthermore, ten healthy males were administered 319 mg per day of liposomal or non-liposomal lactoferrin for four weeks, and immune status was monitored at 0, 1 and 4 weeks after the intake as well as three weeks after stopping it. In this double-blinded comparative study, the IFN-alpha producibility was significantly increased only in the liposomal lactoferrin group during administration and decreased 3 weeks after stopping it, while the IFN-alpha producibility was unchanged in the non-liposomal lactoferrin group. Although the biological mechanism of IFN-alpha producibility enforced by liposomal lactoferrin has not been wholly understood, it is suggested to be a novel active constituent having preventive and therapeutic effects on inflammatory diseases, cancer and infectious diseases such as chronic hepatitis C.     

IL-12 gene therapy is an effective therapeutic strategy for hepatocellular carcinoma in immunosuppressed mice

Immunosuppressive therapy for organ transplantation is essential for controlling rejection. When liver transplantation is performed as a therapy for hepatocellular carcinoma (HCC), recurrent HCC is one of the most fatal complications. In this study, we show that intratumoral murine IL-12 (mIL-12) gene therapy has the potential to be an effective treatment for malignancies under immunosuppression. C3H mice (H-2(k)), injected with FK506 (3 mg/kg) i.p., were s.c. implanted with 2.5 x 10(6) MH134 cells (H-2(k)) and we treated the established HCC with electroporation-mediated gene therapy using mIL-12 plasmid DNA. Intratumoral gene transfer of mIL-12 elevated intratumoral mIL-12, IFN-gamma, and IFN-gamma-inducible protein-10, significantly reduced the number of microvessels and inhibited the growth of HCC, compared with HCC-transferred control pCAGGS plasmid. The inhibition of tumor growth in immunosuppressed mice was comparable with that of mIL-12 gene therapy in immunocompetent mice. Intratumoral mIL-12 gene therapy enhanced lymphocytic infiltration into the tumor and elicited the MH134-specific CTL response even under FK506. The dose of FK506 was sufficient to prevent the rejection of distant allogenic skin grafts (BALB/c mice, H-2(d)) and tumors, B7-p815 (H-2(d)) used as transplants, during mIL-12 gene therapy against MH134. Ab-mediated depletion studies suggested that the inhibition of tumor growth, neovascularization, and spontaneous lung metastasis by mIL-12 was dependent almost entirely on NK cells and partially on T cells. These results suggest that intratumoral mIL-12 gene therapy is a potent effective strategy not only to treat recurrences of HCC in liver transplantation, but also to treat solid malignant tumors in immunosuppressed patients with transplanted organ.     

Lysyl oxidase and MMP-2 expression in dehydroepiandrosterone-induced polycystic ovary in rats

Polycystic ovary syndrome (PCOS) is characterized by cystogenesis; however, the cause of this cystogenesis is unknown. At ovulation, preovulatory collagenolytic activities in the ovarian follicles increase and various proteinases are needed to degrade the tissues surrounding the follicles. To clarify the roles of enzymes in collagen degradation of the follicular wall of polycystic ovary (PCO) in relation to the cystogenesis, we examined expression of lysyl oxidase (LOX), which initiates cross-link formation of the collagen and elastin in the extracellular matrix, and expression of matrix metalloproteinases (MMPs) in ovaries of model rats with PCO induced by dehydroepiandrosterone (DHEA) compared with MMP expression in control rats. DHEA treatment increased LOX mRNA expression to more than three times the control value (P: < 0.01). MMP-2 mRNA expression in control rats was threefold greater than that in the DHEA-induced group (P: < 0.05). Expression of both latent and active forms of MMP-2 in controls was more than twice that in the DHEA-induced group (P: < 0.05) as shown by Western blotting, and expression of the active form of MMP-2 was also twice as high in the controls as in the DHEA-treated group (P: < 0.05) as shown by zymography. Our results suggest that depression of MMP-2 activity and increased LOX expression may be one of the causes of the cystogenesis of PCO.