Comparison of characteristics between F344 and Slc:Wistar rats--Slc:Wistar rats cannot be distinguished from the F344 strain

With respect to F344/DuCrj and Slc: Wistar rats, both widely used in Japan, it was found that there is a close similarity in the changes of body weights and survival rates, and in the organ distribution and incidence of spontaneous tumors. To examine the degree of homozygosity between F344 and Slc: Wistar strains, tumor transplantation and skin grafting were performed. The bladder carcinomas that originated from F344/DuCrj rats grew subcutaneously in the other F344 strains and Slc: Wistar rats, but did not grow in the other Wistar-derived strains. The skin grafts between F344/DuCrj or F344/NSlc and Slc: Wistar rats were accepted, but those between F344/DuCrj or Slc: Wistar and the other Wistar-derived strains were rejected. These results suggest that Slc: Wistar rats cannot be distinguished genetically from the F344 strain of rats.     

Direct up-regulation of estrogen receptor by triiodothyronine in rat pituitary tumor MtT/F84.

To investigate a possible effect of triiodothyronine (T3) on the regulation of estrogen receptor, estrogen dependent rat pituitary tumor, MtT/F84, was studied in rats which received surgical thyroidectomy (Tx) or were given propylthiouracil (PTU) and were supplemented with T3. In T3 loaded rats, the grafted tumor showed high estrogen receptor levels (160-200% of control), whereas low estrogen receptor levels (20-35% of control) were observed in the tumors grown in Tx and PTU treated rats. A single injection of T3 to Tx rats with MtT/F84 increased the estrogen receptor level in a time dependent manner and reached the maximal level at 6 h. In primary culture of MtT/F84 cells, T3 increased the specific 3H-estrogen binding to tumor cells in a dose dependent manner (165% of control by 10(-7)M T3) and also in a time dependent (maximum at 12 h) manner. These data suggest that T3 directly up-regulates the estrogen receptor level in MtT/F84 cells.     

The kinetics of YOYO-1 intercalation into single molecules of double-stranded DNA

Recent studies have suggested that treatment of glucocorticoid to immature growth hormone (GH)-producing cell line, MtT/S cells, dramatically induced the accumulation of GH-containing secretory granules in the cytosol and differentiated into mature GH-producing cells. However, the molecular mechanism of glucocorticoid-induced GH-containing secretory granule biogenesis in the MtT/S cells remains unknown. In the present study, we found that GH mRNA expression was facilitated by application of glucocorticoid. We artificially increased GH synthesis by transfection of green fluorescent protein-tagged GH (GH-GFP) gene. We found that the artificial elevation of GH expression in the cells did not accumulate the secretory granules in the cytosol, whereas glucocorticoid-induced the biogenesis of granules in GH-GFP-expressing MtT/S cells. We next performed DNA microarray and real-time RT-PCR analysis and found that glucocorticoid significantly altered the expression of membrane trafficking-related protein, syntaxin11 (Syx11). Immunocytochemical analysis further demonstrated that Syx11 positive structures were well colocalized with GH-containing granules in both MtT/S cells and rat anterior pituitary gland. Our findings indicate that glucocorticoid regulate the expression of Syx11 and facilitate the biogenesis and the trafficking of GH-containing granules in the MtT/S cells.     

Antigen presentation by Langerhans cells in vivo: donor-derived Ia+ Langerhans cells are required for induction of delayed-type hypersensitivity but not for cytotoxic T lymphocyte responses to alloantigens

T cell activation in response to allogeneic stimulation and hapten-specific delayed-contact hypersensitivity responses in vivo can be initiated by Ia-bearing epidermal Langerhans cells (LC). By using a murine heterotopic corneal allograft model, we have investigated the requirement for allogeneic LC as antigen-presenting cells (APC) in the in vivo induction of delayed-type hypersensitivity (DTH) and cytolytic T lymphocyte (CTL) responses to alloantigens in fully allogeneic and H-2 I region-disparate strain combinations. LC-deficient, avascular central corneal allografts from BALB/c donors failed to induce DTH responsiveness when grafted to a subdermal bed on C57BL/6 recipients (p greater than 0.05), yet antigen-specific primary CTL reactivity developed within 7 days after grafting. LC-containing corneal-limbus allografts or central corneal allografts containing a latex bead-induced infiltrate of LC resulted in intense DTH as well as CTL responsiveness when grafted in this same strain combination. Similarly, LC-containing but not LC-deficient corneal allografts from A.TL donors induced DTH responsiveness in I region-disparate A.TH hosts despite the fact that these grafts survived for prolonged duration (less than 28 days). By contrast, CTL induction in I region-disparate hosts was independent of the presence of allogeneic LC. Corneal epithelial cells of grafts removed from I region-disparate hosts 7 days posttransplantation were shown by immunohistology to express the Iak antigens of donor origin. The possibility that bone marrow-derived allogeneic LC were a sufficient requirement for DTH induction was confirmed in experiments performed with CB6F1----B6 bone marrow chimeras used as corneal allograft donors. Corneal-limbus grafts obtained from mice 90 days after chimerization were shown by immunohistology to contain Iad-bearing CB6F1 LC as a sole source of class II alloantigens. When grafted to C57BL/6 recipients, LC-containing chimeric corneas induced DTH responsiveness that was similar in magnitude to that observed in C57BL/6 mice grafted with chimeric skin, yet no DTH response to LC-deficient chimeric central corneal grafts was observed. Moreover, in all cases, the chimeric corneal and skin allografts survived for prolonged duration (greater than 28 days). These results demonstrate that donor-derived LC act as APC in the induction of DTH responsiveness to allogeneic tissue; however, there was no apparent requirement for allogeneic LC in the induction of CTL responses to class I or class II MHC alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitating the frequency of initiation and cH-ras mutation in in situ N-methyl-N-nitrosourea-exposed rat mammary gland

Mammary carcinogenesis is a multistep process consisting minimally of initiation and promotion/progression stages. The rate-limiting stage in the carcinogenesis process is undetermined but can in part be addressed by estimating the frequency of initiation, a heritable early event. Here, we use an in vivo limiting dilution transplantation assay to estimate initiation frequency in a rat mammary epithelial stem-like cell population that was exposed in situ to 50 mg/kg N-methyl-N-nitrosourea (NMU) administered i.v. We estimate that this dose resulted in the killing of 65% of exposed mammary cells. Known numbers of cells surviving NMU exposure were grafted into fat-pads of recipient rats in which the cells grew and differentiated into structurally and functionally normal mammary glands. Recipient rats were hormonally manipulated to provide maximal promotion of initiated cells. Mammary carcinomas developing at graft sites were quantitated over a 2-year period. Based on these results, we estimate that at least 1 surviving NMU-exposed mammary cell in 7,200 was initiated. Seventeen % of these graft site carcinomas had an activated H-ras oncogene with a G to A mutation in codon 12. This suggests that at least 1 mammary cell in 43,000 was mutated in this fashion by in situ exposure to NMU. These data suggest that cH-ras represents approximately 1 of 5 of the initiation events produced by NMU exposure of rat mammary glands.     

Stem cell characteristics of transplanted rat mammary clonogens

Rat mammary glands contain a subpopulation of clonogenic epithelial cells with large proliferation and differentiation potentials. When transplanted, the clonogens in monodispersed rat mammary epithelial cell suspensions give rise to either alveolar units (AUs) or ductal units (DUs) depending on the nature of the hormonal milieu in the graft recipient. Clonogens are also the primary cells of origin of mammary cancer following exposure to ionizing radiation or chemical carcinogens. Given the other stem cell characteristics of mammary clonogens, it would be expected that the primary AUs and DUs to which they give rise when grafted and hormonally stimulated (a) would be derived from the same clonogenic cell subpopulation, (b) would contain all of the functionally differentiated cell types of homologous parts of comparably stimulated mammary glands in situ, and (c) would also contain clonogen subpopulations capable when subtransplanted of giving rise to secondary AUs and DUs of similar cell composition. The current experiments were designed to test these expectations. The data are discussed in the context of results of previous studies with this and other experimental models. The results further support the conclusion that rat mammary clonogens are multipotent mammary stem cells.     

Establishment in culture of a multihormone-secreting cell strain derived from the MtT/F4 rat pituitary tumor.

A clonal cell strain F4C1 has been established from the transplantable rat pituitary tumor MtT/F4 and has been maintained in continuous culture for two years. The cells grow with a population doubling time of 48 hours; the karyotype with a modal number of 39 chromosomes includes a pair of large metacentric marker chromosomes. F4C1 cells in culture produce growth hormone and prolactin but not adrenocorticotropin in contrast to the MtT/F4 tumor which secretes all three hormones in the host rat. The cloned cells lack specific receptors for thyrotropin-releasing hormone and do not respond to this agent with increased prolactin or decreased growth hormone production. Treatment with hydrocortisone results in a small increase in growth hormone and a small decrease in prolactin production. Tumors generated in rats from injected F4C1 cells secrete prolactin and growth hormone but not adrenocorticotropin. The results suggest that growth hormone and prolactin are produced by a single cell type in the MtT/F4 tumor.     

Strong and weak immune responses across the same major histocompatibility barrier in rats

Inbred rat strains, Fischer 344 (F-344) and Lewis (LEW), share the serologicalAg-Bl allele and react very weakly in mixed lymphocyte culture (MLC). Despite this apparent identity atAg-B, these strains differ markedly in their immune responses to anAg-B disparate third strain Marshall 520 (M-520) (Ag-B6). F-344 recipients allowed M-520 heart grafts an extended survival, whereas LEW recipients rejected them rapidly. F-344 and M-520 showed a weak response in MLC in contrast to a strong response for LEW and M-520. F-344 produced antisera in response to injection of M-520 cells that had a relatively high antibody titer but low cytotoxic activity. F-344 responded to another strain, Buffalo (BUF) (alsoAg-B6), in a similar fashion. F-344 apparently can produce a strong allogeneic response, as it was able to rapidly reject heart grafts from (LEW x Brown-Norway) F₁ donors (LBN) (Ag-B 1/3). The low response of F-344 to M-520 probably was not due to shared antigens between the two strains because M-520 heart grafts underwent rapid rejection in LEW hosts highly tolerant to F-344. To explain the contrasting response of F-344 and LEW to theAg-B6 disparity, we propose that it is controlled by an immune-response gene(s); that F-344 has a low-responding allele and LEW has a high-responding allele. The data do not reveal a location for this proposed gene. The high-responding allele appears to be dominant, as M-520 hearts were rejected rapidly by (F-344 x LEW) F₁ recipients.     

Successful cardiac allografts in syngeneic radiation chimeras

Summary Successful cardiac allografts were accomplished across the major histocompatibility complex of rats. LEW and F344 (Ag-B ²) rats were lethally irradiated and grafted with WF (Ag-B ¹) hearts on day 0. Either on day 0 or day 2, the hosts were repopulated with syngeneic hemopoietic cells. The best results were obtained (86%) when a mixture of 3.0 × 10⁷ non-adherent syngeneic bone marrow and thymus cells were used to repopulate the recipients. In contrast, all of the WF to LEW heart grafts were rejected within 30 days if syngeneic thoracic duct and bone marrow cells were used to repopulate the host.Supported by Grant HL18186 from the National Institutes of HealthRecipient of a Research Career Development Award CA70879 from the National Institutes of Health     

Production of H-Y Antibody by Female Mice that Fail to Reject Male Skin

WHEN inbred mice are grafted with skin from inbred donors that differ from the recipients only by a single minor histocompatibility antigen, it is commonly observed that some recipients will retain their skin grafts while others will reject them. This is true of incompatibility for H-Y antigen, which is responsible for the rejection of male grafts by otherwise histocompatible inbred females of the same inbred strain¹. Thus in the DBA/2 (DBA) strain, male-to-female skin grafts are rejected by only some recipients; in the C57BL (B6) strain, females always reject male skin; and C3H/An (C3H) females usually accept male skin grafts indefinitely.     

Binding of glucocorticoid receptors to mammary chromatin acceptor sites

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.     

Pseudophosphorylated prolactin (S179D PRL) inhibits growth and promotes beta-casein gene expression in the rat mammary gland

We have investigated the individual roles of unmodified prolactin (U-PRL) and a mimic of phosphorylated PRL (S179D PRL) in mammary development. Recombinant versions of the PRLs were delivered to rats throughout pregnancy at a rate of 6 microg/24 h per rat and to non-pregnant females at a rate of 24 microg/24 h per rat. Measurement of progesterone, corticosterone, and estradiol showed no effect of the administered PRLs on the levels of these other mammotropic hormones. Histological and morphometric analysis showed U-PRL to cause mammary growth, whereas S179D PRL inhibited growth. Molecular analysis demonstrated decreased beta-casein expression in the mammary glands of the U-PRL-treated animals at term and increased beta-casein expression in the mammary glands of the S179D PRL-treated animals. Superior beta-casein gene expression in response to S179D PRL versus U-PRL was confirmed in HC11 cells. We conclude that U-PRL is important for growth, whereas S179D PRL promotes at least one measure of differentiated function in the mammary gland.     

In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action.

We have investigated the mechanisms by which dexamethasone (a synthetic glucocorticoid) stimulates the production of mouse mammary tumor virus (MMTV) by cell cultures derived from mammary carcinomas of GR mice. Treatment of these cells with dexamethasone stimulates a rapid accumulation of intracellular virus-specific RNA which is dependent upon RNA synthesis but not upon DNA or protein synthesis. The effect of dexamethasone is probably mediated by a specific and saturable glucocorticoid receptor. We conclude that the accumulation of MMTV RNA is a primary response to dexamethasone and that the rate of synthesis of MMTV RNA is probably accelerated by treatment with dexamethasone.     

The mechanism of liver splanchnomegaly produced by a transplantable pituitary mammotropic tumor in rats.

The mechanism of liver splanchnomegaly developed in rats bearing a transplantable pituitary mammotropic tumor which secretes large amounts of ACTH and prolactin has been studied. The results indicate that in the first phase of tumor growth liver enlargement could be due to hypertrophy, and later mainly to hyperplasia which overcomes hypertrophy. The adrenal glands were found to play an essential role in the process of liver splanchnomegaly because adrenalectomy prevented the disproportional growth of the liver. Evidence is presented showing that glucocorticoids are the dominant hormones responsible for the development of liver splanchnomegaly.     

Bone marrow-derived T lymphocytes responsible for allograft rejection

Lethally irradiated mice reconstituted with syngeneic bone marrow cells were grafted with allogeneic skin grafts 6-7 weeks after irradiation and reconstitution. Mice with intact thymuses rejected the grafts whereas the mice thymectomized before irradiation and reconstitution did not. Thymectomized irradiated mice (TIR mice) reconstituted with bone marrow cells from donors immune to the allografts rejected the grafts. Bone marrow cells from immunized donors, pretreated with Thy 1.2 antibody and C', did not confer immunity to TIR recipients. To determine the number of T lymphocytes necessary for the transfer of immunity by bone marrow cells from immunized donors, thymectomized irradiated mice were reconstituted with nonimmune bone marrow cells treated with Thy 1.2 antibody and C' and with various numbers of splenic T lymphocytes from nonimmune and immune donors. Allogeneic skin graft rejection was obtained with 10(6) nonimmune or 10(4) immune T cells. The effect of immune T cells was specific: i.e., immune T cells accelerated only rejection of the relevant skin grafts whereas against a third-party skin grafts acted as normal T lymphocytes.     

Rat interstrain antibody response and crossidiotypic specificity

Interstrain analysis of the humoral response of rats to streptococcal group A carbohydrate (SACHO) 1, employing seven inbred strains representing six histocompatibility haplotypes at the Ag-B locus, suggests that the immune response genes to SACHO are not linked to the major rat histocompatibility locus. The low-precipitin response of all seven inbred rat strains was similar to the precipitin response of F₇ Sprague-Dawley rats selectively bred for a low-precipitin response to SACHO. Although strain differences were not apparent in the magnitude of the precipitin response to SACHO, the qualitative expression of anti-SACHO antibodies with restricted heterogeneity was more frequently observed in the August strain of rats than in the six other inbred strains examined. Cross-idiotypic specificity was demonstrated for anti-SACHO antisera obtained from nine inbred rat strains. The observations on idiotypy favor the importance of germ-line genes coding for rat antibody variable region determinants in response to SACHO.In this paper, the following abbreviations are used SACHO streptococcal group A carbohydrate - Aug August 2887 - W/Fu Wistar Furth - M520 Marshall 520 - Cop Copenhagen - F344 Fisher 344 - Buf Buffalo/Cr - BN Brown Norway - GASV group A streptococcal vaccine - DEAE diethylaminoethyl-cellulose - RIA radioimmunoassay     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.     

Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization

These studies address the hypothesis that Ag-bearing epidermal Langerhans cells migrate to the regional lymph node during contact sensitization and function as APC. Skin from C3H mice was grafted onto BALB/c nude mice, and 7 or 14 days later, the recipients were sensitized with FITC through the grafts. APC from lymph nodes draining the site of sensitization were capable of sensitizing C3H recipients to FITC. Because sensitization is MHC restricted, only cells reaching the lymph node from the grafted skin could have induced contact hypersensitivity in C3H mice. Examination of the FITC+ draining lymph node cells by immunofluorescence and immunoelectron microscopy demonstrated that all were Ia+, most were F4/80+, and some contained Birbeck granules. These studies demonstrate that Ia+, FITC+ cells from the skin, at least some of which are Langerhans cells, leave the skin after epicutaneous sensitization with FITC and participate in the initiation of the contact hypersensitivity response within the regional lymph node.     

Induction of transplantation tolerance in guinea pigs by spleen allografts. II. Responses in the mixed leukocyte reaction correlate with the tolerant state

We have developed a model for the induction of transplantation tolerance in the guinea pig by vascularized spleen allografts. Spleen allografts from strain 13 to strain 2 hosts frequently survived in healthy recipients without clinical GVHD or induced clinical GVHD. (2 x 13)F1 to strain 2 spleen allografts survived indefinitely without inducing GVHD. In contrast, strain 2 spleen allografts were rejected by strain 13 hosts. An excellent correlation was observed between the clinical course and the degree of reactivity to donor strain stimulator cells in the MLR. Animals that had rejected their grafts had normal or enhanced proliferative responses in the MLR. Strain 2 hosts with long-term surviving strain 13 or (2 x 13)F1 grafts had markedly suppressed anti-13 responses. Animals with GVHD had a suppressed MLR toward donor strain stimulator cells with simultaneous reactivity to host strain stimulator cells. Cells capable of suppressing the response of normal host strain cells to donor strain stimulators were present in some long-term surviving animals and may be responsible in part for the maintenance of the tolerant state.