Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice

Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitary-gonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5'-ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3') encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymus-pituitary-gonadal axis.     

Spontaneous tumors in nude mice: effect of the viable yellow gene.

The incidence and type of spontaneous tumors in athymic nude (nu/nu) mice (partially inbred in CBA/H background) which were also carrying the viable yellow gene (Avy, derived from C57BL/6JAvy mice), were comparable to those observed in the phenotypically normal nu/+ and +/+ control crosses carrying the Avy gene. The Avy gene increases the incidence of spontaneous and induced tumors in most mouse strains. These results would argue against the thymus dependency of the putative immunological surveillance mechanisms.     

Effects of opioid peptides on the cellular immunity in spleen cells from intact nude mice or nude mice bearing human ovarian carcinoma

Summary The present study was designed to explore the effects of opioid peptides on the lytic activity of spleen cells from intact nude mice or nude mice bearing human ovarian cancer cells (KF). When the spleen cells from intact nude mice were incubated with various concentrations of opioid peptides, the ability of the spleen cells to lyse the KF cells was significantly stimulated between 0.05 nM and 50 nM concentrations of all opioid peptides used in this study. The degree of stimulation was most marked at 5 nM opioid peptides and the most marked stimulatory effect was obtained by -endorphin. On the other hand, the lytic activity of spleen cells from nude mice challenged with the KF cells was about two-fold higher than that of intact nude mice, suggesting that spleen cells from nude mice challenged with KF cells have KF-cell-specific cytotoxicity. Even if the spleen cells were incubated with any concentration of -endorphin or [Met]enkephalin indicated, the lytic activity remained unchanged. In contrast, only -endorphin resulted in a significant increase of the lytic activity between 0.5 nM and 50 nM. These results suggest that opioid peptides play a crucial role in immune surveillance mechanisms.     

Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

Effects of methotrexate-carcinoembryonic-antigen-antibody immunoconjugates on GW-39 human tumors in nude mice

Summary Methotrexate (MTX) was conjugated to an anti-carcinoembryonic antigen monoclonal antibody (NP2) by using amino-dextran as an intermediate carrier. The drug was chemically linked to amino-dextran (averageM _r = 40000), and the resulting MTX-dextran was then site-specifically attached to the carbohydrate moiety of the antibody. Athymic nude mice that carried human colonic GW-39 tumors (s. c.) were treated with the immunoconjugate. In this study, the specific conjugate caused a greater inhibition of the tumor growth than either free MTX or its conjugate with dextran and an irrelevant antibody. The intermediate MTX-dextran and the unlinked mixture of MTX-dextran with NP2 were both relatively ineffective in inhibiting tumor growth. The greatly reduced host toxicity permitted the use of the MTX-dextran-NP2 in a high-dose therapy of this tumor system.Supported in part by U.S.P.H.S. grant CA39 841 from the NIH     

Use of athymic nude mice for in vivo studies of human growth-hormone-secreting pituitary adenomas.

Human growth hormone (hGH)-secreting pituitary adenoma tissue of 31 acromegalic patients was transplanted subcutaneously onto 291 athymic nude mice. 37% of the transplanted adenoma fragments could be maintained vital up to 46 days. Histological examinations of the transplants revealed neither alterations in their morphological characteristics nor signs of growth. A maintenance or linear decline of hGH secretion of the transplants related to their vitality was observed by hGH radioimmunoassay. Estimation of graft vitality was improved by GH-releasing hormone (GHRH) stimulation in regular intervals. The rate of pituitary adenomas responding to GHRH was as high as in a major collective of acromegalic patients. Our method of positive selection of vital xenotransplanted hGH-secreting pituitary adenomas via hGH detection at regular intervals in combination with GHRH stimulation gives the opportunity of reliable in vivo research with these tumors.     

Early multipotential pituitary focal hyperplasia in the alpha-subunit of glycoprotein hormone-driven pituitary tumor-transforming gene transgenic mice

Pituitary tumor-transforming gene (PTTG), a securin protein isolated from pituitary tumor cell lines, is highly expressed in invasive tumors and exhibits characteristics of a transforming gene. To determine the role of PTTG in pituitary tumorigenesis, transgenic human PTTG1 was targeted to the mouse pituitary using the alpha-subunit of glycoprotein hormone. Males showed plurihormonal focal pituitary transgene expression with LH-, TSH-, and, unexpectedly, also GH-cell focal hyperplasia and adenoma, associated with increased serum LH, GH, testosterone, and/or IGF-I levels. MRI revealed both pituitary and prostate enlargement at 9-12 months. Urinary obstruction caused by prostatic hyperplasia and seminal vesicle hyperplasia, with renal tract inflammation, resulted in death by 10 months in some animals. Pituitary PTTG expression results in plurihormonal hyperplasia and hormone-secreting microadenomas with profound peripheral growth-stimulatory effects on the prostate and urinary tract. These results provide evidence for early pituitary plasticity, whereby PTTG overexpression results in a phenotype switch in early pituitary stem cells and promotes differentiated polyhormonal cell focal expansion.     

Use of nude mice in experimental neutron capture therapy with 10B-BPA

Mouse B16 melanoma allografts in nude mice were successfully treated by thermal neutron irradiation after IP injection of 10B-paraboronophenylalanine hydrochloride. The tumor growth was significantly suppressed for 4 weeks after irradiation, compared with animals given neutron irradiation alone. Tumor-bearing nude mice were shown to be useful for evaluating the treatment for melanoma.     

Effects of colchicine on the morphology and prolactin secretion of rat anterior pituitary cells in monolayer culture

The effects of incubation of rat anterior pituitary cells in monolayer culture with 10(-6) M colchicine have been investigated during time-intervals extending from 1 to 96 hours. Prolactin release, as measured by radioimmunoassay, was rapidly inhibited by colchicine, this inhibition being accompanied by increased cellular prolactin content for up to 24 hours of treatment and followed by decreased values of cellular prolactin concentration at later time-intervals. Immunocytochemical localization showed an increased positive reaction for prolactin up to 24 hours after colchicine treatment, whereas transmission electron microscopy demonstrated, in parallel, an increased number of intracellular prolactin secretory granules during the same interval. Longer periods of treatment (24-96 hours) resulted in the appearance of more lysosomes, autophagic vacuoles and microfilaments in the cells, whereas the number of Golgi elements was decreased. Following four hours of colchicine treatment and at later stages, microtubules could no longer be observed in the sections. Scanning electron microscopic data showed that colchicine treatment induced dramatic changes in the cell surface morphology: at short time intervals (4 and 8 hours), the number of microvilli decreased and the cell surface became folded, whereas, later, "bleb"-like protrusions of variable dimensions partially covered the cell surface and seemed to be released from it. These data show a good correlation between secretory activity of prolactin-producing cells and morphological changes induced by colchicine treatment.     

Occurrence and morphology of tumors induced in nude mice transplanted with chrysotile-transformed rat pleural mesothelial cells

Rat pleural mesothelial cells treated in vitro with chrysotile fibers have been successfully transplanted into nude mice. Three cultures (1 untreated, 2 treated) were injected at passage 75; a fourth culture was obtained from a mesothelioma induced in rat by chrysotile fibers. Overall, tumors grew in each series, but the delay between cell injection and tumor formation was 22 wk with untreated cells whereas only 1 or 2 wk were needed with treated cells, and 1 wk with cells from in vivo-induced mesothelioma. Pathological study by light and electron microscopy of tumors is reported here and showed the mesothelial nature of the cells. Comparison between the ultrastructure of the injected cells and tumor cells indicated that the morphology of injected cells was retained in tumors even if the delay in tumor formation was long. These results suggest that this model is useful for investigating mesothelial cell transformation resulting from in vitro or in vivo exposure to certain carcinogens.     

Effects of thymus grafts in nude mice transplated with human malignant tumors

The effect of neonatal thymus grafts implanted in nude mice previously transplanted with three different human malignant tumors - an adenocarcinoma of the colon, a malignant melanoma and a Burkitt's lymphoma - were studied. In all immunologically reconstituted animals tumord were rejected. Tumor rejection stated 2-3 weeks after thymus implantation, and was completed after 30-6 weeks. Histological examination of lymphoid tissues showed a correlation between immunological reconstitution and tumor rejection. The rejection process showed a characteristic histologic picture with 3 phases - an early, an intermediate and a late phase - these were similar in the three tumor types examined. The possible mechanisms of reconstitution and tumor rejection are discussed.     

Complementation of the anti-TNP PFC response of N:NIH nude mice from nude dams by spleen cells of nude mice from heterozygous dams.

Type I and Type II nude (nu/nu) mice are borne and raised by nu/nu dams and +/nu dams, respectively, under SPF conditions. Splenocytes from Type I nude mice exhibit even smaller in vitro PFC responses to trinitrophenylated rabbit erythrocytes than splenocytes from Type II nude mice. Type II splenocytes complement the magnitude of the PFC response of Type I splenocytes. The helper cell-like activity contained in Type II splenocyte suspensions was increased by treating Type II nude donor mice with dibutyryl cyclic AMP, but was essentially unaffected by pretreatment of Type II donors with endotoxin (LPS). The helper-like activity of Type II splenocytes was suppressed by treatment with a rabbit antiserum (plus C′) raised against a mixture of neonatal mouse thymocytes (RAM-T). We interpret the data to indicate that Type II splenocytes contain a larger number of residual T cells and/or a more fully developed population of T precursor cells that initiate or that are capable of being induced to initiate a helper function.     

Effects of selenium deficiency on sperm morphology and spermatocyte chromosomes in mice

Sperm morphology and spermatocyte chromosomes were examined in mice maintained on a Torula yeast diet for 5 weeks. In the selenium-deficient group, the proportion of abnormal sperm was high, ranging from 6.8% to 49.6%, while in the control group it ranged from only 4.0% to 15.0%. The most frequently occurring abnormalities in sperm shape were in the sperm head. There was also a tendency for abnormalities in other regions (neck, midpiece and tail) to be increased. However, in metaphase-I spermatocytes, the frequencies of various types of abnormal chromosomes (univalent chromosomes, translocations and structural anomalies) did not differ between the selenium-deficient and control groups. These findings indicate that selenium may be an essential constituent for spermatogenesis in mice.     

Acute effects of ultraviolet light B on morphology and epidermal cell kinetics in human skin transplanted to nude mice

Grafts of human skin on nude mice were irradiated with varying doses of ultraviolet light B and at various intervals were subjected to histological examination and determination of the epidermal 3H-thymidine labelling index. The studies showed that the human skin in the foreign milieu of the nude mouse retained its ability to respond to phototoxic damage. The findings suggest that the nude mouse/human skin model could be a valuable tool in human photodermatological research.