Pharmacological characterization of the leukocyte kinetics after intranasal antigen challenge in a guinea pig model of allergic rhinitis.

OBJECTIVE AND DESIGN: We characterized the leukocyte kinetics after antigen challenge, and investigated the effects of the thromboxane (TX) A2 antagonist seratrodast, the peptide leukotriene (p-LT) antagonist pranlukast, the antihistaminic drug terfenadine and the glucocorticoid dexamethasone on this leukocyte response in a guinea pig model of allergic rhinitis. SUBJECTS: Male Hartley guinea pigs were used. TREATMENT: Intranasally sensitized guinea pigs were challenged once every week for 15 weeks by inhalation of Japanese cedar pollen as the antigen. Dexamethasone and other agents were administered orally 3 and 1 h, respectively, before the 15th challenge. METHODS: The time-related changes in the numbers of differential leukocytes in nasal cavity lavage fluid (NCLF) and in peripheral blood after pollen inhalation challenge were investigated. The effects of the drugs on the antigen-induced changes in the leukocyte counts were evaluated. In addition, histopathological examination of the nasal mucosa was also performed 5 h after the challenge. RESULTS: There was a marked increase in the number of leukocytes in NCLF, especially of eosinophils, which peaked at 5 h, after antigen challenge in this model. This response was also accompanied by the peripheral blood eosinophilia and neutrophilia. Seratrodast (30 mg/kg), pranlukast (30 mg/kg) and dexamethasone (10 mg/kg) inhibited the eosinophilia in all of the blood, the nasal mucosa and NCLF seen 5 h after the antigen challenge. Terfenadine (10 mg/kg) had no apparent effect on the blood and the mucosal eosinophilia, although it tended to suppress the eosinophil accumulation in NCLF. CONCLUSIONS: These results suggest that the present model is useful for analyzing the mechanisms of antigen-induced eosinophilic inflammation in allergic rhinitis and that both TXA2 and p-LTs, but not histamine, contribute to the antigen-induced eosinophilia in this model of allergic rhinitis.

A competitive enzyme-linked immunosorbent assay-like method for the measurement of urinary hyaluronan.

A highly sensitive method for measurement of urinary hyaluronan with a minimum molecular mass of 2,000 Da was developed without using HPLC or radioisotopes. This competitive enzyme-linked immunosorbent assay-like method, used competitive binding of free hyaluronan in the sample and biotin-labeled standard hyaluronan to hyaluronan binding protein in solid phase. A total of 150 healthy individuals from both sexes at ages from 0 to 100 years was examined by the established method. Hyaluronan of 384 +/- 80 ng/mg creatinine (mean +/- SD) was constantly excreted into urine of 24-40-year-old healthy adults. The urinary hyaluronan levels were significantly higher before age 1 (p < 0.001) and rather high after 90 years compared to the other groups. The average molecular weight of urinary hyaluronan (5,500 Da) was constant through all generations. Sex difference of urinary hyaluronan was not observed both quantitatively or qualitatively.

Periodontal inflammatory and cystlike lesions in BDF1 and B6C3F1 mice.

Periodontal inflammatory and cystlike lesions were detected in aged BDF1 and B6C3F1 mice. Lesions were unilateral, single, and located in the labial region of the incisor teeth. The largest cystlike lesion deformed the skull. Histologically, the cystlike lesions were classified into three types: predominantly an inflammatory reaction, predominantly a cyst formation, and a mixed lesion. These periodontal inflammatory and cystlike lesions were pathogenetically similar and were considered sequential changes; the lesions showing both cyst formation and inflammatory reaction seemed to be intermediate along a path toward formation of large cysts. The cysts probably were derived from periodontal tissue and developed as a result of inflammatory reaction to foreign bodies, including hair. The term "murine periodontal cyst" is proposed for this lesion. Detailed pathogenetic studies on murine odontogenic cystic lesions may be warranted.

Spontaneous lesions of nasal cavity in aging F344 rats and BDF1 mice.

The outline of spontaneous lesions of the nasal cavity that were commonly observed in F344 rats and BDF1 mice used as untreated control groups in ten 2-year carcinogenicity studies were presented. In rats, the common spontaneous lesions were eosinophilic change of the respiratory epithelium and the olfactory epithelium, respiratory metaplasia of glands and the olfactory epithelium, foreign body inflammation, deposit of calcium, and thrombus. In mice, the common spontaneous lesions were eosinophilic change of the respiratory epithelium and olfactory epithelium, and respiratory metaplasia of glands and the olfactory epithelium. Some of these lesions revealed either species or sex-related differences of incidence.

Domain structure of heparan sulfates from bovine organs.

Samples of heparan sulfate, isolated from bovine aorta, lung, intestine, and kidney, were degraded by digestion with a mixture of heparitinases or by treatment with nitrous acid, with or without previous N-deacetylation. Analysis of the resulting oligosaccharides showed that the various heparan sulfate samples all contained regions of up to 8 or 9 consecutive N-acetylated glucosamine residues, as well as contiguous N-sulfated sequences. L-Iduronic acid accounted for a remarkably constant proportion, 50-60%, of the total hexuronic acid units within the latter structures. Of the total iduronic acid units, 36-55% were located outside the contiguous N-sulfated regions, presumably in sequences composed of alternating N-acetylated and N-sulfated disaccharide residues. While most of the iduronic acid units within the N-sulfated blocks were 2-O-sulfated, those located outside were almost exclusively nonsulfated. The heparan sulfate preparations differed markedly with regard to the content of 6-O-sulfated glucosamine units, more than half of which were located outside the N-sulfated block regions. These findings suggest that the formation of iduronic acid residues and their subsequent 2-O-sulfation are coupled within but not outside the contiguous N-sulfated regions of the heparan sulfate chains and, furthermore, that the 2-O- and 6-O-sulfotransferase reactions are differentially regulated during heparan sulfate biosynthesis.

Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a potent angiogenic mitogen. To elucidate the effect of bFGF inhibitors in vivo, anti-bFGF immunoneutralizing monoclonal antibody was prepared. One monoclonal antibody against human bFGF, obtained by cell fusion and designated 3H3, completely inhibited bFGF-induced proliferation of human umbilical vein endothelial cells at a concentration of 100 ng/ml. 3H3 did not bind to acidic fibroblast growth factor or HST1 protein, indicating high specificity for bFGF. Furthermore, the immunoneutralizing activity of 3H3 was examined in vivo. K1000 cells (a BALB/c 3T3 transformant in which the leader sequence-fused bFGF gene was transfected) were transplanted s.c. into BALB/c nude mice. Growth of the tumor cells was inhibited by i.v. treatment with 3H3 at a concentration of 200 micrograms/mouse. Histological observation showed that the antitumor effect of 3H3 was due to the inhibition of bFGF-induced angiogenesis. This experiment provides direct causal evidence for the hypothesis that tumor growth is angiogenesis dependent. This finding could also have implications for the development of novel therapeutic approaches to angiogenic solid tumors.

Antitumor effect of recombinant human interleukin-2 on the growth of murine hemangioendothelioma D14 in nude mice: occurrence of large granular cells in the tumor.

The antitumor effect of recombinant human interleukin-2 (rIL-2) on murine hemangioendothelioma D14 (D14) in female BALB/c-nu/nu mice was examined histologically. D14 cells which had been maintained in vitro were transplanted subcutaneously into nude mice on day 0 (1 x 10(7) cells/mouse). The mice with established tumor on day 28 received rIL-2 subcutaneously at a dose of 20 micrograms/mouse/day for 35 days. On day 63, the mice were killed, and the tumor, spleen and bone marrow were examined histologically. In the mice that had received rIL-2, tumor growth was significantly suppressed. Histologically, there was marked infiltration of large granular cells (about 15-30 microns in diameter) in the tumors. In the adjacent areas, there was a significant increase in the number of tumor cells showing karyorrhexis. The large granular cells (LGC) contained periodic acid Schiff-positive round granules in the cytoplasm and were stained positively for Thy-1.2 surface antigen. The LGC were also positive for asialo GM1 surface antigen but not for Lyt-1, Lyt-2 or IgG surface antigens. This evidence suggests that the LGC are lymphokine-activated killer-like cells which were derived from a natural killer cell lineage. The concomitant increases in the number of LGC and the number of cells showing karyorrhexis in the tumors of the mice treated with rIL-2 suggest that LGC play an important role in the destruction of tumor cells.

Histological studies on the therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) in an experimental endometriosis model in rats.

The therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) on experimental endometriosis in female rats was examined histologically. Endometriosis was produced in rats by autotransplantation of endometrial tissue obtained from the left uterine horn into the renal subcapsular space. In the nontreated rats, the transplants were well established and had formed large cysts containing fluid. The walls of the cysts were composed of epithelium and stroma resembling that of normal endometrium. In the rats which received the microspheres of leuprorelin acetate, growth of the transplant was markedly suppressed as evidenced by the reduced size of the cystic cavity and the flattened and pyknotic epithelium. Also, the uterine and ovarian weight decreased significantly. In the ovariectomized rats, growth of the transplant was also markedly suppressed, and the uterine weight decreased. The present results clearly indicate that a single injection of the sustained-release microspheres of leuprorelin acetate markedly suppresses growth of the transplant and produces uterine and ovarian atrophy in the rats.

Combination therapy of colon carcinoma 26 in mice with recombinant human interleukin-2 and interferon-alpha A/D: occurrence of large granular cells in the tumor.

The antitumor effects of recombinant human interleukin-2 (rIL-2), in combination with recombinant human interferon-alpha A/D hybrid (rIFN-alpha A/D) on colon carcinoma 26 (colon 26) in mice were examined histologically. Colon 26 was transplanted subcutaneously into female BALB/c mice on day 0. The mice bearing the tumor received intramuscular injections of rIL-2, rIFN-alpha A/D or the combination of rIL-2 and rIFN-alpha A/D for 2-10 consecutive days starting on day 7. Mice were killed on days 9, 13, 17 and 21. After day 13, growth of the tumor was significantly suppressed in the mice treated with rIL-2 or rIFN-alpha A/D alone and was stopped in the mice treated with rIL-2 in combination with rIFN-alpha A/D. Histologically, tumor necrosis developed in all treated groups, though the degree was the most severe in the group receiving combination treatment. Many large cells (about 15-30 microns in diameter) infiltrated into the tumor, and they had Thy-1 surface antigen and many periodic acid-Schiff-positive round granules in the cytoplasm. The incidence of these large granular cells was correlated well with the reduction in tumor weight. The ultrastructural features of the large granular cells were very similar to those of murine large granular lymphocyte-like cells maintained in vitro in an IL-2-containing medium. The present large granular cells appear to be a kind of activated lymphoid cells.

Sequential observations of thymic lymphoma development induced in 10-week-old F344 rats by N-propyl-N-nitrosourea.

N-Propyl-N-nitrosourea (PNU) is a strong carcinogen which induces thymic lymphoma and other tumors in F344 rats. In the present experiment, sequential changes of the thymus, spleen, bone marrow, and other organs were examined histologically in F344 rats which were continuously given PNU in the drinking water. Hematopoietic organs, such as the bone marrow, spleen, and thymus rapidly became hypoplastic. Atrophy of the thymus was followed by repopulation and hyperplasia. The latter was characterized by lymphoblast-like cells and was followed by development of early stage lymphoma, which arose from the 10th experimental week onwards. At first, thymic lymphoma was observed to involve thymic lobes unilaterally, later spreading to bilateral lobes, and at the 16th experimental week, metastatic foci were evident in the bone marrow of one rat. In contrast, hypoplasia of the bone marrow continued until the end of the experiment, while hypoplasia of the red splenic pulp continued until the 12th experimental week. These results indicate that PNU-induced lymphomas arise from within the thymus, although it is not possible to rule out a role for the bone marrow as including target cells of PNU. This PNU-thymic lymphoma system in F344 rats should serve as a good model for the study of experimentally induced thymic lymphoma.

Development of thymic lymphomas by oral administration of N-nitroso-N-propylurea and establishment of transplantable lines of thymic lymphoma in F344 rats.

Forty-eight female F344/DuCrj rats were given a 400-ppm solution of N-nitroso-N-propylurea (CAS: 816-57-9) continuously in their drinking water. Thymic lymphomas were induced most frequently (85%) followed by duodenal tumors (48%). Sixteen tumors were examined by the immunofluorescence inhibition test for murine leukemia virus-related antigens; 15 were negative and the other was weakly positive. Twenty-six tumors were intraperitoneally and subcutaneously transplanted syngeneically; 25 (96%) were successfully transplanted intraperitoneally and 24 (92%) subcutaneously. The serial intraperitoneal transplantation was continued, and 22 lines of transplantable lymphoma in an ascites form were established. In almost all tumor lines, tumor cells took in a high percentage of recipient rats and caused the death of the recipients within 12-23 days. The thymus, liver, spleen, greater omentum, and lymph nodes were frequently invaded by transplanted tumor cells. The tumor lines were considered to be of T-cell lineage.

Desensitization of gonadotropin-releasing response following vaginal consecutive administration of leuprolide in rats.

The vaginal absorption of a potent luteinizing hormone-releasing hormone analog (leuprolide), and the gonadotropins (luteinizing hormone and follicule-stimulating hormone)-releasing response after continuous vaginal and subcutaneous administration of the analog to rats were determined by the radioimmunoassay. A marked suppressive effect on the gonadotropin-releasing response along with long-lasting serum levels of leuprolide were paradoxically observed after consecutive 3-d vaginal administration of the analog at doses of 1 microgram/kg or greater. Pituitary function recovered progressively with cessation of the treatment but was not complete 4 d after cessation. Continuous vaginal and subcutaneous infusion resulted in an almost complete inhibition of both gonadotropins response. It is suggested that effective desensitization of the pituitary gonadotropin response is elicited by continuous administration of leuprolide and, therefore, the vaginal administration resulting in prolonged serum levels of the analog could be preferable as a self-administration method for medical treatments such as anti-tumor therapy and birth control.

Regression of rat mammary tumors by a potent luteinizing hormone-releasing hormone analogue (leuprolide) administered vaginally.

Potent luteinizing hormone-releasing hormone analogues are known to cause regression of hormone-dependent mammary tumors. We have observed that high and long-lasting serum levels of a potent luteinizing hormone-releasing hormone analogue [desglycyl10-(D-leucyl6) luteinizing hormone-releasing hormone ethylamide, leuprolide] resulted from vaginal administration which effectively caused down regulation in the pituitary by chronic treatment. Regression of 7,12-dimethylbenz(a)-anthracene-induced mammary tumors in Sprague-Dawley rats by consecutive daily vaginal administration of leuprolide was investigated. In untreated rats, 71% of tumors were growing at 8 weeks, whereas after i.p. injection of leuprolide (500 micrograms/kg) all tumors were regressing 2 weeks after commencement of treatment and 86.7% of tumors disappeared by 8 weeks. Vaginal administration of 100 micrograms/kg for 8 weeks produced regression in 80% of tumors and disappearance in 35%. The vaginal administration of a higher dose (500 to 5000 micrograms/kg) produced highly significant antitumor effects [regression in 82.2 +/- 4.0% (S.E.) and disappearance in 52.9 +/- 2.1%]. These results are consistent with the effects produced by ovariectomy. Whereas 13 and 7 new tumors appeared in untreated rats and those treated vaginally with leuprolide (100 micrograms/kg), respectively, only one or two tumors appeared in i.p. and vaginally (above 500 micrograms/kg) treated rats during treatment. Histological classification of the mammary tumors after treatment indicated therapeutic effects similar to those shown by tumor size determination. Thus, it was concluded that vaginal application of leuprolide at doses above 500 micrograms/kg might be a potentially useful method for antitumor therapy.