The C57BL/6 nude, beige mouse: a model of combined T cell and NK effector cell immunodeficiency

This article describes the construction and establishment of a double congenic nude, beige C57BL/6 (B6 nu, bg) mouse strain. The mice do not show higher fragility than C57BL/6 nude mice and the double congenic strain can be maintained under conventional mouse housing conditions. Although the B6 nu, bg display a very low natural killer activity which cannot be enhanced by an interferon inducer (poly(I-C], they lack responsiveness to a T cell mitogen (concanavalin A); and they also show extremely low responsiveness to a B cell mitogen (0128: B12 Escherichia coli lipopolysaccharide) probably as a result of combined effects of the beige and nude genes in the C57BL/6 genetic context.     

Karyotype and heterochromatin pattern of the field mouse,Apodemus argenteus Temminck

The field mouse,Apodemus argenteus Temminck, has 46 chromosomes. The autosomes comprise 20 pairs of acrocentrics and 2 pairs of metacentrics. The X chromosome is represented by an outstandingly large submetacentric element, while the Y is an acrocentric corresponding in size to the 5th or 6th pair of autosomes. All of the autosomes and gonosomes can be unequivocally identified by their characteristic Q-band or G-band patterns. The constitutive heterochromatin, as revealed by C-banding, is localized at the centromeric regions of all autosomes, the short arm and the proximal 1/3 of the long arm of the X chromosome, and the entire Y chromosome. The C-band-positive segments which constitute 33.5% of the genome exhibit dark fluorescence after Q-banding, late DNA replication, faint or positive staining reaction to G-banding, fast reassociation of DNA revealed by AO staining, and allocyclic behavior of the sex-bivalent in male meiosis. An exception to the above is the distal segment of the Y which is positive to both C- and Q-banding. The giant X chromosome occupies 13.1% of the genome, leaving 5.6% of euchromatic segments, the latter value being equivalent to that of the original type X.     

Carcinogen metabolism in human skin grafted onto athymic nude mice: a model system for the study of human skin carcinogenesis

Human skin grafted onto athymic nude mice maintains its major histological features and may provide a useful system with which to assess the carcinogen interaction with human skin. Significant differences were observed in basal levels of cytochrome P-450 and cytochrome P-448-dependent monooxygenase activities between human grafted and nude mouse epidermis. Topical application of crude coal tar (CCT) to human skin transplanted onto nude mice resulted in 3.9 & 3.5; 3.2 & 2.9 and 1.1 & 1.2 fold increases in mouse and human epidermal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin deethylase (ERD) and ethoxycoumarin deethylase (ECD) activities, respectively. CCT applied topically to mouse skin resulted in 27.8 & 6.4; 12.8 & 3.3 and 1.7 & 2.6 fold increases in mouse and human epidermal AHH, ERD and ECD activities, respectively. Topical application of coal tar either onto human transplanted skin or to mouse skin also resulted in substantial induction of hepatic and pulmonary AHH and ERD activities. These studies indicate that human skin grafted onto nude mice preserves its metabolic capacity and offers a useful model system with which to assess the effects of polycyclic aromatic hydrocarbons and CCT on cutaneous xenobiotic metabolism in the human population.     

Karyomorphology and GC-rich heterochromatin pattern in Passiflora (Passifloraceae) wild species from Decaloba and Passiflora subgenera

Thirteen wild species of Passiflora were analyzed using conventional and CMA/DA/DAPI staining to evaluate the karyotype diversity between and within the subgenus Decaloba and Passiflora. The karyotypic features indicate that both subgenera have a conserved chromosome number, as reported before for several species. Submetacentric (sm) chromosomes were found in species from both subgenera, suggesting that sm chromosomes are not restricted to a particular subgenus. The analysis of the karyotypic heterogeneity enabled to distribute the species in three groups, but with no support to phylogenetic and taxonomic levels. The application of fluorochromes allowed for the visualization of CMA⁺/DAPI⁻ blocks, which in our studies always correlated with the occurrence of satellites, showing that occurrence of two chromosome pairs with satellites per cell is a characteristic shared by some species from both subgenera. This feature does not always have relationship with the basic chromosome number. The data found in this study will help to understand the phylogeny, cytotaxonomy, and evolution of the genus Passiflora showing that karyotypic variation can be seen between and within the subgenus Decaloba and Passiflora.     

Role of reactive nitrogen species in development of hepatic injury in a C57bl/6 mouse model of human granulocytic anaplasmosis

Human granulocytic anaplasmosis (HGA), caused by the granulocytic rickettsia-like organism Anaplasma phagocytophilum, is the 3rd most frequent vector-borne infection in North America. To understand the disease mechanisms of HGA, we developed a murine model that lacks clinical disease yet exhibits characteristic histopathologic and immunologic changes. Because the degree of hepatic histopathology is unrelated to high bacterial numbers, tissue injury in HGA is thought to occur due to products of innate immunity, such as nitric oxide (NO) and reactive nitrogen species (RNS) from cytokine-activated macrophages. To test the hypothesis that RNS cause hepatic tissue damage, mice received either water treated with a nonspecific inhibitor of inducible nitric oxide synthase, L-NAME, or untreated water for 7 to 10 d before infection and continuing thereafter. Mice were euthanized for tissue harvest at 0, 7, 14, or 21 d after infection to assess differences in histopathology, hepatic bacterial load, RNS quantity in urine and liver, and serum chemistry values. Overall, L-NAME treatment had a beneficial effect, resulting in lower histopathology scores and RNS levels compared with those of untreated mice. There were no significant differences in hepatic bacterial load among treatment groups of infected mice. The observed increases in serum glucose and alanine aminotransferase levels on day 14 appear to be unexpected side effects of L-NAME administration. HGA is best characterized as an immunopathologic disease rather than one caused by direct bacterial injury to the host. Therefore, human and animal patients with HGA likely would benefit from therapy targeting reduced inflammation to supplement anti-infective modalities.     

Tumor lipids and liver lipid metabolism in the model human lung carcinoma/nude mice

Tumor lipids were studied in the experimental model Human Lung Carcinoma/nude mice as well as the effect of this human neoplasm on the host liver lipid metabolism. Fatty acid profiles from tumoral lipids revealed the loss of specificity for fatty acid composition in triglycerides. Host liver fatty acid composition and cholesterol metabolism were affected by the implanted human lung tissue. A noticeable increase ratio between saturated/unsaturated fatty acids was observed in host liver fatty acid phospholipids (1.17 +/- 0.17) in comparison to control liver (0.84 +/- 0.04). Cholesterol synthesis was assessed "in vivo" by means of [14C]acetate incorporation. The specific radioactivity of [14C] cholesterol was increased by a factor of about 6 in host liver as compared with control liver. This observation along with the marked decrease in the cholesterol content of host liver and the hypocholesterolemia detected in the host mice led us to suggest an increase in the liver cholesterol catabolism promoted by the presence of the tumor.     

Cell fusion of human and mouse cells as a source for new cells retaining human markers. Analysis of DNA content, membrane and cytoplasmic antigen expression.

Flow cytometry and ultrastructural morphometry were used to study some characteristics of cells obtained by fusion with polyethylene-glycol 4000 between mouse fibroblasts 3T3.4E and normal keratinocytes (3T3.4E x NHK) or hand wart human keratinocytes (3T3.4E x HWK), at late passages. The cell cycle and the expression of human beta 2-microglobulin, human EGF-receptors (EGF-r), vimentin were simultaneously studied by flow cytometry. Epithelial CaSki cells, derived from a human uterine carcinoma, expressing high levels of beta 2-microglobulin, EGF-r and vimentin, were used as a positive control. In mouse fibroblasts 3T3.4E only vimentin was expressed whereas in cells derived from fusion, human beta 2-microglobulin, human EGF-r and vimentin were detected. The cell cycle analysis revealed that the peak position of G0/G1 differed with the cells (channel 11 for 3T3.4E cells, 13 for 3T3.4E x HWK and 15 for 3T3.4E x NHK). The area of the cell compartments from each cell type was also different by quantitative ultrastructural morphometry. The hybrid phenotype was maintained in late passages in cells (3T3.4E x NHK) and (3T3.4E x HWK), as shown by the expression of human antigens, differences in DNA contents and nuclear area. Flow cytometry may be a very accurate and precise tool for studying low antigenic expression. The combination of different methods including analysis of DNA content, antigenic expression and ultrastructural morphometry confirmed that 3T3.4E, 3T3.4E x NHK and 3T3.4E x HWK cells are different cell types. These techniques are complementary to cell phenotype analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionizing radiation as an initiator in the mouse two-stage model of skin tumor formation

The initiating potential of a range (7.5 to 22.5 Gy) of 4 MeV X rays was studied using the mouse skin two-stage model of carcinogenesis. A single dose of radiation was followed by 60 weeks of promotion with 12-O-tetradecanoyl phorbol-13-acetate (TPA) (8 nmol, two times per week). Since the proliferative state of a target cell population is known to influence carcinogen-induced tumorigenesis, we also investigated the effect of TPA on tumor incidence when applied as a single dose (17 nmol) 24 h prior to irradiation. Evidence presented here indicates that ionizing radiation can act as an initiator in this model system. All groups of animals that were promoted with TPA developed papillomas regardless of radiation treatment; however, only those groups of animals that received irradiation followed by TPA promotion developed squamous cell carcinomas. The incidence of nonepidermal tumors was similar between all radiation dose groups and was independent of TPA promotion. Our results also indicate that TPA pretreatment prior to irradiation results in an overall increase in the total tumor incidence, including both epidermal and nonepidermal tumors.     

Characterization of the serotoninergic system in the C57BL/6 mouse skin.

We showed expression of the tryptophan hydroxylase gene and of tryptophan hydroxylase protein immunoreactivity in mouse skin and skin cells. Extracts from skin and melanocyte samples acetylated serotonin to N-acetylserotonin and tryptamine to N-acetyltryptamine. A different enzyme from arylalkylamine N-acetyltransferase mediated this reaction, as this gene was defective in the C57BL6 mouse, coding predominantly for a protein without enzymatic activity. Serotonin (but not tryptamine) acetylation varied according to hair cycle phase and anatomic location. Serotonin was also metabolized to 5-hydroxytryptophol and 5-hydroxyindole acetic acid, probably through stepwise transformation catalyzed by monoamine oxidase, aldehyde dehydrogenase and aldehyde reductase. Activity of the melatonin-forming enzyme hydroxyindole-O-methyltransferase was notably below detectable levels in all samples of mouse corporal skin, although it was detectable at low levels in the ears and in Cloudman melanoma (derived from the DBA/2 J mouse strain). In conclusion, mouse skin has the molecular and biochemical apparatus necessary to produce and metabolize serotonin and N-acetylserotonin, and its activity is determined by topography, physiological status of the skin, cell type and mouse strain.     

Combined resveratrol, quercetin, and catechin treatment reduces breast tumor growth in a nude mouse model

Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 microM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 microM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression.     

Synergy between the T3/antigen receptor complex and Tp44 in the activation of human T cells

In addition to the T3/antigen receptor complex (T3/Ti), other T cell surface molecules participate in early events involved in human T cell activation. In this report we document that monoclonal antibody 9.3, which recognizes a 90,000 dalton homodimer expressed on human T cells, synergizes with ligands reacting with T3/Ti to activate purified T cells and Jurkat, a human T cell leukemic line. Unlike phorbol myristate acetate (PMA), 9.3 was able to synergize only with anti-T3 or anti-Ti if these antibodies were immobilized. Moreover, 9.3 failed to synergize with the calcium ionophore ionomycin. At high concentrations only, 9.3 could synergize with PMA in the activation of Jurkat and a T3/Ti negative mutant of Jurkat. At such high concentrations of 9.3, small transient increases in cytoplasmic free calcium ((Ca++)i) were detected in quin 2-loaded Jurkat cells. This increase in (Ca++)i was the result of release of internal stores of calcium. 9.3 induced the hydrolysis of polyphosphoinositides, albeit the magnitude of inositol phosphates generated in response to 9.3 was substantially less than that observed with anti-Ti. No effect on pkC translocation was observed in Jurkat cells stimulated with 9.3. Although the small increase in (Ca++)i induced by 9.3 may account for its synergy with PMA, this effect is unlikely to account for the more potent synergistic effect observed with 9.3 and phytohemagglutinin or immobilized anti-T3 and anti-Ti antibodies.     

Study of lactoferrin gene expression in human and mouse adipose tissue,human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers

Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.     

Reactivity to SV40 T antigen in athymic (nude), anti-thymocyte serum-treated, and normal mice.

Antisera prepared in syngeneic mice by hyperimmunization with intact SV40-transformed mouse cells or with somatic cell hybrids between SV40-transformed human and normal mouse cells exhibit anti-SV40 tumor (T) antigen reactivity. Athymic mice bearing tumors formed by SV40-transformed mouse, human or mouse-human hybrids were not reactive with SV40 T antigen. Anti-thymocyte serum (ATS)-treated mice also lacked T antigen reactivity during suppressive treatment but developed antibody to T antigen after discontinuing ATS treatment and tumor regression. We conclude that that presence of growing tumors in the mouse is not necessary for the production of anti-SV40 T antigen antibodies but that helper thymus-derived cells are essential for the humoral response.     

Endogenous DNA damage clusters in human skin, 3-D model, and cultured skin cells

Clustered damages-two or more oxidized bases, abasic sites, or strand breaks on opposing DNA strands within a few helical turns-are formed in DNA by ionizing radiation. Clusters are difficult for cells to repair and thus pose significant challenges to genomic integrity. Although endogenous clusters were found in some permanent human cell lines, it was not known if clusters accumulated in human tissues or primary cells. Using high-sensitivity gel electrophoresis, electronic imaging, and number average length analysis, we determined endogenous cluster levels in DNA from human skin, a 3-D skin model, and primary cultured skin cells. DNA from dermis and epidermis of human skin contained extremely low levels of endogenous clusters (a few per gigabase). However, cultured skin fibroblasts and keratinocytes-whether in monolayer cultures or in 3-D model skin cultures-accumulated oxidized pyrimidine, oxidized purine, and abasic clusters. The levels of endogenous clusters were decreased by growing cells in the presence of selenium or by increasing cellular levels of Fpg protein, presumably by increasing processing of clustered damages. These results imply that the levels of endogenous clusters can be affected by the cells' external environment and their ability to deal with DNA damage.     

Substitution pattern of the CArG element in human and mouse genomes

cis-Elements CArG bound by serum response factor (SRF) are presently being intensively studied, but little is known about the substitution pattern of functional CArG elements. Here, we have performed the first evolutionary analysis of CArGome in the human and mouse genome through bioinformatic methods and statistical tests. We calculated the substitution rate at each site of the functional CArG elements. The results showed that the core sites of the functional CArG elements evolved faster than did the background DNA, indicating that these sites were likely to evolve under positive selection. Moreover, a strong TATA "motif" was evident in the core region within the functional CArG elements in both human and mouse promoters. This motif could probably be a major contribution to the formation of the spatial structure, which was important for CArG-SRF recognition. Thus, the study further revealed the sequence character and substitution pattern of CArG elements and provided useful information for the study of the SRF-binding efficiencies of CArG promoters in functional assays.     

Blood group A/B-defined glycosyltransferase and A/B blood group antigens in human normal and malignant endometrium in relation to morphology,age and oestrogen levels

We have used monoclonal antibodies to study the expression and regulation of A/B antigens and A/B transferase in normal and malignant human endometrium by immunohistochemistry. Staining was evaluated against blood group status, morphology, age ad serum oestrogen levels. The expression of the antigens, in contrast tothe expression of the transferase, was related to the A subtype (A₁/A₂) and the ABH secretor status. Normal, non-secretory endometria and most well-differentiated endometrial carcinomas from ABH secretors expressed the antigens and the transferase, but showed a morphology-dependent variation in the expression and degree of coexpression. n contrast, most grade 2 and 3 carcinomas were found to lack both structures, whereas secretory endometrium had a high expression of the transferase but expressed the antigens on only a few cells. The transferase expression was correlated inversely with age and positively with the level of free oestradiol in serum. Our findings suggest that A/B antigenic expression in the endometrium may be regulated at different levels — at the A/B transferase level and at a precursor substrate lvel — and that both genetic and hormonal factors are probably involved in the regulatory process.     

The progression in the mouse skin carcinogenesis model correlates with ERK1/2 signaling

BACKGROUND: The ras family of proto-oncogenes encodes for small GTPases that play critical roles in cell-cycle progression and cellular transformation. ERK1/2 MAP kinases are major ras effectors. Tumors in chemically treated mouse skin contain mutations in the Ha-ras proto- oncogene. Amplification and mutation of Ha-ras has been shown to correlate with malignant progression of these tumors. Cell lines isolated from mouse skin tumors represent the stages of tumor development, such as the PDV:PDVC57 cell line pair and B9 squamous carcinoma and A5 spindle cells. PDVC57 cells were selected from PDV cells, which were transformed with dimethyl-benzanthracene (DMBA) in vitro and then transplanted in adult syngeneic mice. The PDV:PDVC57 pair contains ratio of normal:mutant Ha-ras 2:1 and 1:2, respectively. This genetic alteration correlates with more advanced tumorigenic characteristics of PDVC57 compared to PDV. The squamous carcinoma B9 cell clone was isolated from the same primary tumor as A5 spindle cell line. The mutant Ha-ras allele, also present in B9, is amplified and overexpressed in A5 cells. Therefore these cell line pairs represent an in vivo model for studies of Ha-ras and ERK1/2 signaling in mouse tumorigenesis. MATERIALS AND METHODS: The ERK1/2 status in the above mouse cell lines was examined by using various molecular techniques. For the study of the tumorigenic properties and the role of the ras/MEK/ERK1/2 pathway in the cell lines mentioned, phenotypic characteristics, colony formation assay, anchorage-independent growth, and gelatin zymography were assessed, after or without treatment with the MEK inhibitor, PD98059. RESULTS: ERK1/2 phosphorylation was found to be increased in PDVC57 when compared to PDV. This also applies to A5 spindle carcinoma cells when compared to squamous carcinoma and papilloma cells. The above finding was reproduced when transfecting human activated Ha-ras allele into PDV, thus demonstrating that Ha-ras enhances ERK1/2 signaling. To further test whether ERK1/2 activation was required for growth we used the MEK-1 inhibitor, PD98059. The latter inhibited cell proliferation and anchorage-independent growth of squamous and spindle cells. In addition, PD98059 treatment partially reverted the spindle morphology of A5 cells. CONCLUSIONS: These data suggest, for the first time, that oncogenicity and the degree of progression in the mouse skin carcinogenesis model correlates with ERK1/2 signaling.