IL-4/IL-13 antagonist DNA vaccination successfully suppresses Th2 type chronic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic disease with a Th2-type-cytokine dominant profile. Several cytokines and related peptides have been used for the treatment of AD but they were ineffective because of their limited biological half-life. We have recently developed a highly efficient mouse dominant negative interleukin (IL)-4/IL-13 antagonist (IL-4DM), which blocks both IL-4 and IL-13 signal transductions. OBJECTIVE: To examine the effects of IL-4DM in vivo in an AD model induced by the repeated exhibition of oxazolone (OX). METHODS: Plasmid DNA was injected intraperitoneally to cause an experimental AD-like dermatitis. The effect was evaluated by ear thickness, histological findings, and mast cells counts in the inflamed skin. The plasma IgE and histamine levels were measured. Cytokine production in skin and splenocytes were also analysed. RESULTS: Mice treated with control plasmid developed marked dermatitis with mast cells and eosinophil infiltration, and had increased plasma IgE and histamine levels with a Th2 type splenocyte cytokine profile. Treatment with mouse IL-4 DNA augmented the ear swelling and thickness with an increased dermal eosinophil count, plasma histamine level, and production of splenocyte IL-4. However, IL-4DM treatment successfully controlled the dermatitis, decreased the mast cell and eosinophil count, and suppressed plasma IgE and histamine levels. Splenocytes produced an increased level of IFN-gamma. CONCLUSION: These data showed that the simultaneous suppression of IL-4/IL-13 signals successfully controlled Th2-type chronic dermatitis. IL-4DM DNA treatment is a potent therapy for AD and related diseases.

Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice.

Sialoadenectomies performed on 8-week-old female SHN and GR mice markedly reduced the numbers of precancerous and cancerous lesions in their mammary glands that had been mildly hypoplastic; the mice were necropsied when they were 30 weeks old. The success rate of the mammary cancer transplantation to isogenic male SHN or C3H mice was lower in the sialoadenectomized animals, and growth of the grafted tumors was delayed after gland removal. Some tumor development resumed in the hosts that received mouse epidermal growth factor after surgery. Therefore, we believe this growth factor may play a role in the multistage process of mouse mammary carcinogenesis.

Cytokeratin expression in subungual squamous cell carcinoma.

Cytokeratin expression in subungual squamous cell carcinoma was investigated in order to evaluate the origin and state of differentiation of the tumour. The tumour nests contained cytokeratin 14, 16 and 17, which were also expressed in the nail bed. Therefore, cytokeratin expression in subungual squamous cell carcinoma may reflect its indolent clinical prognosis.

Intervention of T-cells in transportation of mouse mammary tumor virus (milk factor) to mammary gland cells in vivo.

Using BALB/c nu/nu, BALB/c nu/nufC3H (BALB/c nu/nu mice raised by C3H/HeN foster mother), BALB/c thymus-engrafted BALB/c nu/nufC3H, BALB/c nu/+, and BALB/c nu/+fC3H mice, we examined what kinds of cells are carriers of blood-borne mouse mammary tumor virus (B-MMTV). A radioimmunoassay and an immunoperoxidase assay revealed the presence of MMTV-gp52 antigen in the mammary glands of all BALB/c nu/+fC3H and BALB/c thymus-engrafted BALB/c nu/nufC3H mice (more than 60 days old) but only of some BALB/c nu/nufC3H mice (more than 120 days old): those that possessed a significant number of functional T-cells. BALB/c nu/+ mice did not show the antigen expression at any age. Transfer experiments of cells or plasma from young (less than 12 weeks) BALB/c nu/nufC3H to BALB/c +/+ virgins revealed that cells besides T-cells can also become carriers of B-MMTV. This was confirmed by Southern blotting analyses; exogenous provirus DNA sequences were found in B-cells as well as T-cells of BALB/c nu/+fC3H mice. However, when young BALB/c nu/nu mice were inoculated with BALB/c nu/nufC3H blood, they did not show the MMTV-gp52 antigen expression. Transfer experiments of purified T-cells, B-cells, natural killer cells, and macrophages from BALB/c fC3H mice to BALB/c nu/nu mice revealed that only T-cells have the ability to transfer viral activity to the mammary glands. These results suggest that B-MMTV is carried from the gastrointestinal tract to the mammary glands by lymphoid cells such as T-cells and B-cells, then transferred to the mammary gland cells by the T-cells.

Modulations of glucocorticoid-induced apoptosis linked to the p53 deletion and to the apoptosis susceptibility gene Rapop1 (Radiation-induced apoptosis 1).

We have analysed the effects of p53 and of the apoptosis susceptibility gene Rapop1 (Radiation-induced apoptosis 1) located on chromosome 16 on glucocorticoid- and radiation-induced in vivo apoptosis of thymocytes. For those analyses, we used Rapop1 semicongenic mice heterozygous for the STS and BALB/cHeA alleles in the chromosomal segment containing Rapop1 in the BALB/cHeA background, mice bearing a p53 deficient allele in the BALB/cHeA background and the genetic crosses between these mice. The p53 wild type mice with a STS/A allele at the Rapop1 locus were less susceptible to both radiation- and glucocorticoid-induced apoptosis than those with homozygous BALB/cHeA alleles at this locus. Surprisingly, glucocorticoid-induced apoptosis was enhanced in the p53 hemizygous mice and considerably increased in the p53 nullizygous mice. In contrast, a sizable reduction of radiation-induced apoptosis was seen in the p53 hemizygous mice. The low susceptiblity to glucocortocoid-induced apoptosis linked to the STS allele of Rapop1 was less pronounced in the p53 hemizygous mice and a diminished effect of Rapop1 on radiation-induced apoptosis was seen in these mice. Although it remains to be established whether the genes modulating glucocortocoid-induced apoptosis are identical to p53 and Rapop1, our data suggest that p53 and Rapop1 may participate in glucocorticoid-induced apoptosis of thymocytes.

Keratinizing squamous epithelium associated with Syringocystadenoma papilliferum differentiates towards infrainfundibulum: case report with immunohistochemical study of cytokeratins.

We describe a case of Syringocystadenoma papilliferum (SCAP) with keratinizing squamous epithelium in a 26-year-old female presenting with a dark brown to black nodule on her forehead. After surgical excision, the specimen was examined immunohistochemically using antibodies against cytokeratin (CK) 1, 8, 10, 14, 17, 18 and 19. Within the keratinizing squamous epithelium, CK1, 10, 14 and 17 were present, whereas the other CKs were absent. Based on CK expression, keratinizing squamous epithelium in SCAP seems to differentiate towards the infrainfundibulum.

Induction of mammary carcinomas by the direct application of crystalline N-methyl-N-nitrosourea onto rat mammary gland.

The effectiveness of the direct application of crystalline N-methyl-N-nitrosourea (MNU) onto the mammary gland was compared with the systemic intraperitoneal (i.p.) administration method for the induction of mammary carcinomas in female Sprague-Dawley (S-D) rats. The effectiveness was also tested in genetically resistant female Copenhagen (Cop) rats. The 10 mg crystalline MNU was dusted directly onto the right-inguinal mammary gland, or 50 mg/kg body weight MNU solution was given i.p. at 50 days of age. Animals were palpated for tumor detection twice weekly and killed when the tumor reached 1-2 cm in diameter or were necropsied 30 weeks after carcinogen treatment. In S-D rats, all of the 78 tumors produced by dusting were adenocarcinomas. By contrast, 40 tumors produced i.p. were adenocarcinomas, 1 was fibroadenoma, and 5 were lactating adenomas. The cumulative incidence of mammary carcinoma was high in the dusting and the i.p. groups (12/12; 100% and 11/13; 84%, respectively). However, the dusting groups showed a high number of carcinoma per rats (6.5 vs. 3.6; P < 0.01) and short cancer latency (13.8 weeks v.s. 28.1 weeks; P < 0.001) than the i.p. groups. In Cop rats, although low (4/11; 36%), adenocarcinomas were developed by the dusting method. In both strains, adenocarcinomas displayed various degrees of differentiation but no evidence was found for metastasis. For MNU-administration, the direct dusting technique is an effective method and offers added advantages of ease for the induction of mammary carcinomas in rats.

Morphological detection of cell kinetics and progesterone receptor expression during growth and regression of pregnancy-dependent mammary tumors of GRS/A mice.

Cell kinetics (cell proliferation and cell death) and the expression of progesterone receptor (PgR) during growth and regression of pregnancy-dependent mammary tumors (PDMT) of female GRS/A mice were investigated on the histologic level. Cell proliferation was determined using monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (PC10) and cell death was detected using the TUNEL method. PgR expression was determined using monoclonal anti-PgR antibody (10A9). In growing PDMT (type P tumor) obtained during days 16-20 of pregnancy, numerous PCNA labeling was observed in both the epithelial and mesenchymal cells, whereas PgR was found only in epithelial cells and no TUNEL signal was detected. In regressing PDMT obtained during days 0-5 of lactation, the level of PCNA labeling was low and the PgR-positive cells were preferentially labeled by the TUNEL staining, which led to microcyst formation (cystic degeneration). Pale cell carcinoma was shown to be pregnancy-dependent, since the tumor cells were universally PgR-positive, and TUNEL-positive signal with low PCNA-labeling was detected after the delivery.

Colon carcinogenesis in shrews by intrarectal infusion of N-methyl-N-nitrosourea.

Intrarectal infusion of a 0.15 ml solution containing 1.5 mg or 0.5 mg of N-methyl-N-nitrosourea (MNU) was given to female shrews from 6 weeks of age. Fifteen shrews were given 16 doses of 1.5 mg MNU administered biweekly (group 1), 15 shrews were given 24 doses of 0.5 mg MNU weekly (group 2), and four untreated shrews served as controls (group 3). Moribund shrews were killed during the course of the experiment and all remaining animals were killed at 37 weeks of age and terminated the experiment. The mean age when killed was 30.8 weeks in group 1 and 36.2 weeks in group 2. All autopsied shrews in group 1 and group 2, 13 shrews each, had colon cancers, and there were a few small intestinal cancers and uterine squamous cell carcinomas, while no tumors were seen in the untreated shrews. The colonic lesions were of both exophytic and endophytic type with a variety of histologies and depths of invasion. Mesenteric lymph node metastasis was seen in 31% (4/13) of group 1 and 23% (3/13) of group 2. Thus, MNU, a colonotrophic carcinogen in rodents, also induced colon cancer in shrews in the present study.

Influence of perinatal genistein exposure on the development of MNU-induced mammary carcinoma in female Sprague-Dawley rats.

Genistein, a phytoestrogen, was subcutaneously (s.c.) injected to pregnant Sprague-Dawley CD rats on gestational days 16-20 at either 25 mg (Group 1) or 5 mg/day (Group 2). Female offspring of mothers not exposed to genistein during pregnancy received 12.5 mg genistein s.c. at neonatal days 15 and 18 (Group 3), or received vehicle only (Group 4). At 35 days of age, 4-9 female offspring from each group were autopsied to observe the influence of genistein, and remainder of female offspring received 50 mg/kg N-methyl-N-nitrosourea (MNU) intraperitoneally and were sacrificed when mammary tumors were larger than 1 cm in size or when they reached 35 weeks of age. Genistein treatment during the perinatal period resulted in lower body weight and lower relative uterine-ovarian weight at 35 days, and a prolonged estrus cycle with a long estrus phase at 12-16 weeks. However, at 35 days (time at MNU administration), mammary gland development, cell proliferation rate (PCNA labeling index), and the number of estrogen receptor (ER)- and progesterone receptor (PgR)-positive cells were similar between genistein-treated and untreated rats. Twenty-five or 5 mg genistein/day in utero (between days 16 and 20 of gestation) or 12.5 mg genistein/day on neonatal days 15 and 18 did not affect the incidence of mammary tumors > 1 cm or the latency but did increase the number of mammary cancer lesions when MNU was administered at the time when the mammary gland growth in genistein-treated and untreated rats was similar. Thus, perinatal genistein is an endocrine disrupter and increases the multiplicity of MNU-induced mammary carcinoma in rats.

Establishment of DDD/1-Mtv-2/Mtv-2, nu/nu and DDD/1-Mtv-2/Mtv-2, nu/+ mice: preliminary characterization in relation to MTV antigen expression and mammary tumorigenesis.

To investigate the effects of the T-cell deprivation on viral mammary tumorigenesis, two double congenic mouse strains of the DDD genetic background, DDD/1-Mtv-2/Mtv-2, nu/nu and DDD/1-Mtv-2/Mtv-2, nu/+, were produced by the cross between DDD/1-Mtv-2/Mtv-2 (DDD-Mtv-2) and DDD/1-nu/nu mice, followed by repeated intercross breedings. Expression of the mouse mammary tumor virus (MTV)-gp52 antigen was demonstrated in the mammary glands of mice from 14 days on, in both -nu/nu and -nu/+ females. Mammary gland development was comparable in both strains, but, the incidence of mammary cancer was lower in the T-cell-deprived mice.

Alleviation of renal disease and lymphadenopathy in MRL-Fasp(lrcg)/Fas(lprcg) (MR-lpr(cg)) mice neonatally infected with mouse mammary tumor virus encoding superantigen strongly reactive with TCR Vbeta8.2 element.

Mouse mammary tumor virus transmitted by FM mice (FM-MMTV) encodes a superantigen (SAg) characterized by strong reactivity with TCR Vbeta8.2 element and broad spectrum of Vbeta reactivity. To investigate what effects the expression in vivo of FM-MMTV SAg exhibits on the course of the disease in a lupus-prone model, MRL/MpJ-Fas(lprcg)/Fas(lprcg) (MRL-lpr9cg) mice, neonatally FM-MMTV-infected MRL-lprcg(MMTV) and uninfected MRL-lpr(cg) mice were compared for various disease parameters. In MRL-lprcg(MMTV), survival was significantly prolonged, glomerulonephritis, proteinuria, and lymphadenopathy were clearly ameliorated, and the production of serum immunoglobulin G (IgG), complement-activating IgG2a, and cryogenic IgG3 autoantibodies, which are thought to be pathogenic to kidneys, and circulating immune complexes (IC), and glomerular IC deposition were significantly suppressed. FM-MMTV infection deleted Vbeta8.2+ cells by about 90% and Vbeta14+ cells less efficiently in all of the CD4+, CD8+, and B220+ CD4- CD8- or double-negative (DN) T-cell populations, and Vbeta8.1+ cells in the CD4+ population but not in the others. Similar deletion profiles of CD8+ and DN T cells support that DN T cells are derived from the CD8 lineage. The results imply that the specific regulation of the immune system with viral SAg has a potential for development of an attractive immunomodulatory therapy of autoimmune diseases.

Synergistic effect of MNU and DMBA in mammary carcinogenesis and H-ras activation in female Sprague-Dawley rats.

The combined application of N-methyl-N-nitrosourea (MNU) and 7,12-dimethylbenz[alpha]anthracene (DMBA) was compared with the administration of each carcinogen alone as to the effectiveness of the induction of mammary carcinomas and the influence of H-ras oncogene activation in female Sprague-Dawley rats. At 50 days of age, group 1 received 30 mg/kg MNU intraperitoneally (i.p.), group 2 received 30 mg/kg DMBA i.p., group 3 received 60 mg/kg MNU i.p., group 4 received 60 mg/ kg DMBA i.p., group 5 received 30 mg/kg MNU followed by 30 mg/kg DMBA i.p., group 6 received 30 mg/kg MNU i.p. and then 30 mg/kg DMBA intravenously (i.v.) and group 7 remained untreated. Animals were killed when the largest mammary tumor reached 1-2 cm in diameter or were necropsied when they were 30 weeks of age. MNU i.p. produced no deaths (groups 1 and 3), however, the i.p. administration of DMBA induced death due to peritonitis (groups 2, 4 and 5), whereas the i.v. administration of DMBA suppressed the death (group 6). All of the tumors produced by MNU were adenocarcinomas of mammary origin. In contrast, DMBA produced tumors of other than mammary origin. The combined treatment with DMBA and MNU increased the mammary carcinogenic effect; it significantly increased the mean number of mammary cancers per rat. With either carcinogen alone and in combination, the mammary carcinomas produced identical adenocarcinoma histology. Of the mammary carcinomas induced by the combined application of MNU and DMBA (group 6), all 11 tumors from five rats showed the GGA to GAA transitional mutation in H-ras codon 12 (38%) and all 18 tumors from the other 10 rats remained as wild-type. An H-ras point mutation at codon 61 was not detected.

Neoplastic response to dmba powder in mammary and interscapular tissues in wistar furth and copenhagen female rats.

To investigate genetic factors in local tumorigenesis, a dusting of 1 mg of 7,12-dimethylbenz(a)-anthracene (DMBA) powder was directly applied to exposed mammary tissue in 15 Wistar/Furth (WF), 20 Copenhagen (Cop), 19 (WF x Cop) F1, 16 backcross (BC) (F1 x WF) and 19 BC (F1 x Cop) females, at the age of 30 days. By 28 weeks after dusting, gross tumors were induced in 15 WF, 17 Cop, 12 (WF x Cop) F1, 11 BC (F1 x WF) and 14 BC (F1 x Cop) rats. Depending on their neoplastic response, carcinomatous response was 93% in WF, 5% in Cop, 16% in (WF x Cop) F1, 31% in BC (F1 x WF) and 0% in BC (Fl x Cop) rats, and sarcomatous response was 20% in WF, 85% in Cop, 58% in (WF x Cop) F1, 44% in BC (F1 x WF) and 73% in BC (F1 x Cop) rats. When dusting was performed on the interscapular fat tissue of 21 WF and 22 Cop females at 38 days of age, sarcomas were found in 67% of WF and 91% of Cop rats. Therefore, susceptibility and supressor gene(s) are suspected in mammary epithelial cells of the WF and Cop, respectively, for carcinogenesis, while co-dominant allele for sarcomagenesis exists in the Cop and WF mammary stromal cells.

Strain differences in neoplastic response to dmba-induced uterine vascular tumors in mice.

We examined strain differences in neoplastic response to DMBA-induced uterine vascular tumors in mice. Female BALB/c, C57BL, C3H, SWR/J, GRS/A and DDD/1-Mtv-2 mice were treated with a single intraperitoneal injection (ip) of 1 mg of 7,12-dimethylbenz(a)anthracene (DMBA) at 4 weeks of age, and observed until they were 32 weeks of age. The BALB/c, C57BL, C3H strains showed a high incidence of uterine vascular tumors (87%, 93%, and 90%, respectively), while in contrast, the SWR/J, GRS/A, and DDD/1-Mtv-2 strains showed a low incidence (17%, 11% and 15%, respectively); these differences were striking. The mice of American origin (BALB/c, C57BL and C3H) appeared to be susceptible to DMBA, while those of European origin (SWR/J, GRS/A and DDD/1-Mtv-2) were resistant to the induction of uterine vascular tumors.

Induction of apoptosis in head and neck tumor-cell lines by anti-fas antibody.

Programmed cell death, currently termed apoptosis plays a key role in the maintenance of the steady state in continuously renewing tissues. Since little is known of apoptosis in head and neck tumors, we studied morphological changes in head and neck tumor-derived cell lines (KB, KBrc, HSC-2,3,4), induced by anti-Fas antibody. Light and electron microscopic examinations were carried out after culturing these cell lines with the antibody for 1-2 days. The antitumor effect of anti-Fas antibody on tumor cells differed with the cell lines. Most of the cell lines that were sensitive to anti-Pas antibody showed evidence of enhanced apoptosis when the cells were pretreated with interferon-gamma. The results suggest that the strategy of induction of apoptosis by anti-Fas antibody may be considered in treatment of some tumors of head and neck.

Keratin phenotypes in Basal-cell carcinomas of the skin.

Immunohistochemical profiles of 23 cases of basal cell carcinoma (BCC) were examined and compared with adjacent normal skin using three keratin antibodies, 34betaB4, 312C8-1 and E3, which recognize keratin 1, 14 and 17, respectively. In normal skin, 34betaB4 was expressed in suprabasal cells of the epidermis, sebaceous duct cells and the outer root sheaths of hair follicles at the level of sebaceous duct insertion. 312C8-1 was seen in basal cells of the epidermis, the outer root sheaths of hair follicles, and the peripheral cells of sebaceous glands. E3 was detected in the outer root sheaths of hair follicles, and the peripheral cells of sebaceous glands, while it was absent in the epidermis. In BCC, 312C8-1 and E3 were homogeneously found in all 23 cases, and 34betaB4 was sporadically expressed in 3. Therefore. the results suggest that the keratin expression of BCC resembles that of the pilosebaceous apparatus.

Esophageal carcinoma in house musk shrews, Suncus murinus (Insectivora), induced by N-methyl-N'-nitro-N-nitrosoguanidine.

Female 6-week-old shrews were given a solution of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a concentration of 50 micrograms/ml or 100 micrograms/ml in the drinking water. All 11 shrews receiving 100 micrograms/ml MNNG died 8-13 days after the beginning of carcinogen administration and 6 of the 20 shrews receiving 50 micrograms/ml MNNG died after 10-54 days. When animals were between 43 and 54 weeks of age, multiple esophageal lesions were evoked in all 14 that had received 50 micrograms/ml MNNG for 30 weeks. All shrews developed a protruding, ulcerative, or superficial type of squamous-cell carcinoma of the esophagus, accompanied by papillomas. Local invasion was seen in squamous-cell carcinoma but no distant metastasis was noted. None of the 5 control shrews developed any esophageal abnormality. No gastric adenocarcinoma, intestinal sarcoma, or other tumors were induced with MNNG. It can be concluded that MNNG has a carcinogenic effect on shrew esophageal epithelium.

Effects of genistein and synergistic action in combination with eicosapentaenoic acid on the growth of breast cancer cell lines.

Genistein, a prominent isoflavone in soy products, produced dose- and time-dependent in vitro growth inhibition at high concentrations (at least 185 microM) with an IC50 of 7.0-274.2 microM after 72 h incubation in four breast cancer cell lines (DD-762, Sm-MT, MCF-7 and MDA-MB-231) and one breast epithelial cell line (HBL- 100) of human and animal origin; it stimulated estrogen-receptor-positive MCF-7 cells at low concentrations (3.7 nM-37 microM). Genistein-exposed cells underwent apoptosis, confirmed by G2/M arrest followed by the appearance of a sub-G1 fraction in cell-cycle progression, and by a characteristic cell ultrastructure. The apoptosis cascade was due to up-regulation of Bax protein, down-regulation of Bcl-XL protein, and activation of caspase-3. Genistein acted in synergism with eicosapentaenoic acid (EPA), a fish oil component, on human breast cancer MCF-7 cells (genistein > 93.2 microM and EPA > 210.9 microM) and on MDA-MB-231 cells (genistein > 176.1 microM and EPA > 609.3 microM). Dietary intake of genistein in combination with EPA may be beneficial for breast cancer control.