Expression of fetal antigens in fetal and adult cells during long-term culture

Expression of fetal antigens in early and late passages of tissue culture cells derived from C3H/HeN and C57BL/KaLw mouse fetal cells and from lung tissue of young C57BL/6N mice was investigated by the isotopic antiglobulin technique. The late passage lines of fetal cells had undergone "spontaneous" neoplastic transformation in culture. The antisera were produced by syngeneic immunization with 5000 R x-irradiated tissues from C3H/HeN and C57BL/6N fetuses of 1 to 2 weeks gestation. Fetal antigens were found to be retained even after 5 years in cell lines derived from fetal tissues. In these lines no consistent change in fetal antigen expression could be correlated with neoplastic transformation. In contrast, the early passages of adult cells did not have detectable amounts of fetal antigens. However, fetal antigen(s) was demonstrated in cells of the late passages, and cells of both lines grew as sarcomas when next assayed 55 days later. In addition, fetal antigens were also present in established tumor lines in culture.     

Comparative study on high fat diet-induced 4-hydroxy-2E-nonenal adducts in the hippocampal CA1 region of C57BL/6N and C3H/HeN mice

In the present study, we investigated the influences of a high fat diet (HD) fed for 12 weeks, on lipid peroxidation and antioxidant enzyme using 4-hydroxy-2E-nonenal (HNE)-modified proteins (HNE-mp) and Cu,Zn-superoxide dismutase (SOD1) in the hippocampal CA1 region (CA1) in C57BL/6N and C3H/HeN mice. Body weights and body weight gains were significantly higher in HD fed C57BL/6N mice than in low fat diet (LD) fed C57BL/6N and LD or HD fed C3H/HeN mice. In the HD fed C57BL/6N and C3H/HeN mice, HNE-mp immunoreactivity and protein levels were much higher than in the LD fed C57BL/6N or C3H/HeN mice. In particular, HNE-mp immunoreactivity and protein levels in HD fed C57BL/6N mice was higher than that in the HD fed C3H/HeN mice. SOD1 immunoreaction was detected in the non-pyramidal cells of C57BL/6N mice, while in the C3H/HeN mice SOD1 immunoreaction was observed in CA1 pyramidal cells. The SOD1 immunoreactivity in the LD fed C57BL/6N and C3H/HeN mice was slightly, but not significantly decreased compared to that in the HD fed C57BL/6N and C3H/HeN mice, respectively. In addition, ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia in the HD fed C57BL/6N showed hypertrophy of cytoplasm, which is the characteristics of activated microglia. These results suggest that HD fed C57BL/6N mice are more susceptible to lipid peroxidation in the CA1 than in LD fed C57BL/6N and LD or HD fed C3H/HeN mice without any differences of SOD1 expression. In Koo Hwang and Il Yong Kim have contributed equally to this article.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.     

A novel methodology for analysis of cell distribution in chimeric mouse organs using a strain specific antibody

Chimeric animals are very useful for analysis of cell lineage, homeostasis in tissue architecture, and cell-cell interactions during both organogenesis and carcinogenesis. However, there is not a generally effective means for marking cells of chimeric mice. We have therefore developed a polyclonal antibody that is useful for this purpose. This antibody specifically recognizes those cells derived from C3H strain mice. The specificity of this antibody was checked by both immunoblotting and immunoadsorption methods. The antigens were immunohistochemically detected in cytoplasm of both epithelial and mesenchymal cells of C3H/HeN strain mouse in many different organs, but not the corresponding cell types from BALB/c or C57BL/10 or several other mouse strains. The validity of these antibodies as markers for C3H cells was further checked by tissue recombination experiments and in mixed cultures of mouse and rat cells. In each case the antibody recognized only the C3H mouse cells. Next, chimeric mice were prepared between strains C3H/HeN and BALB/c, and C3H/HeN and C57BL/10 mice. Chimeras 2-mo old were examined for antigen distribution using the indirect immunofluorescence method. Many tissues in chimeric mice were composed of cells that were both stained and unstained by the anti-C3H specific antigen. The chimeric patterns were classified into four types, A-D. In well-defined structural units such as intestinal crypts, small intestinal villi, kidney convoluted tubules, exocrine gland acini, ovarian follicles, thyroid gland follicles, stomach glands, adrenal cortex, lingual papillae, etc., (A) each unit was composed entirely of either positive or negative cells, or else (B) in some organs each unit was composed of both types of cells. In the uniform tissues without such distinguishable units, such as stratified squamous epithelium, mesenchymal tissue, corpora lutea, pituitary gland, Islets of Langerhans, adrenal medulla etc., (C) the tissue was composed of definite small cell groups made entirely of either positive or negative cells, or else (D) the tissue was composed of both types of cells which were intermingled with one another. These findings strongly suggest that the chimeric patterns demonstrated here reflect the cell proliferative unit in each tissue. This cell marker system has proven useful for analysis of cell lineage and cell renewal systems in many organs of chimeric mice.     

Association of fertility, fitness and longevity with the murine Ah locus among (C57BL/6N) (C3H/HeN) recombinant inbred lines

The Ah locus encodes a cytosolic receptor which controls the induction of enzymes that metabolize drugs, chemical carcinogens, and other environmental pollutants. B6NXC3N recombinant inbred lines have been developed from the progenitors C57BL/6N and C3H/HeN inbred mouse strains. Ah phenotyping at each generation has resulted in the establishment of some lines containing high levels of the high-affinity Ah receptor; other lines, very low levels. A genetic model involving two unlinked loci is offered to explain the distribution of Ah receptor levels among (C57BL/6N) (C3H/HeN)F2 individuals. Between generations 7 and 13, individual females and males from the B6NXC3N recombinant inbred lines were crossed with DBA/2N males and females. Presence of high levels of the high-affinity Ah receptor in both female and male B6NXC3N mice was found to be associated with greater fertility, fitness, and longer life span. The data suggest that these parameters are correlated with the Ah locus or a closely segregating gene.     

A simple approach for mouse embryonic stem cells isolation and differentiation inducing embryoid body formation

Stem cells were derived from hatched blastocyst-stage mouse embryos of the C57BL/6 strain employing a knockout serum replacement instead of the traditional fetal calf serum, thereby avoiding the use of immunosurgery. Although fetal calf serum was not good for isolation of stem cells, a combination of this serum plus knockout serum increased the expansion rate of the cell culture. The derived cells were capable of maintaining an undifferentiated state during several passages, as demonstrated by the presence of alkaline phosphatase activity, stage-specific embryonic antigen 1 (SSEA-1), and octamer binding protein 4 (Oct-4). Suspension culture in bacteriological dishes gave better results than the hanging drop method for differentiation by means of embryoid body formation. Mouse embryonic stem cells showed spontaneous differentiation into derivatives of the 3 germ layers in culture media supplemented with fetal calf serum but not with knockout serum.     

Activation of murine T cells by toxic shock syndrome toxin-1. The toxin-binding structures expressed on murine accessory cells are MHC class II molecules

Toxic shock syndrome toxin-1 (TSST-1)-binding structures present on murine lymphoid tissues were investigated by using 125I-TSST-1. T-depleted C57BL/6 spleen cells incubated with TSST-1 for 3 h at 0 degree C were mitogenic to splenic T cells, indicating that the former cells bind and present TSST-1 to T cells. TSST-1-binding activity was observed in C57BL/6 splenic B cells and L cells transfected with I-Ab genes, but not in splenic T cells and control L cells. Scatchard plot analysis showed that these B cells and transfectants bound TSST-1 with similar binding affinity. SDS-PAGE analysis showed that lysates of C57BL/6 spleen cells and the I-Ab-positive transfectants contain a single band which bound TSST-1 and comigrated with I-Ab heterodimers. TSST-1-binding activity observed clearly in C57BL/6. BALB/c, and C3H/HeN spleen cells and L cells transfected with I-Ab or I-Ak genes was not reduced by paraformaldehyde fixation. Binding of 125I-TSST-1 to the three spleen cells was markedly reduced by anti-I-A antibodies, but not by anti-I-E antibodies. C57BL/6, C3H/HeN, and (C3H/HeN x C57BL/6) F1 T cells were activated by TSST-1 to proliferate and produce IL-2 in the presence of FT6.2 cells, LT1-30-3 cells and either of them, respectively, but not in the presence of control L cells. These results indicate that I-A molecules function as the structures via that accessory cells directly bind TSST-1 on the cell surface and present a triggering signal of TSST-1 to T cells.     

Lung tumor-reactive cytotoxic lymphocytes generated in mixed lymphocyte reaction between C3HfeB/HeN (H-2kb) and C3H/HeN (H-2k) strain mice.

The tumor-associated transplantation antigen expressed by several transplacentally induced lung tumors of C3HfeB/HeN mice (H-2kb haplotype) has previously been shown to exist as a normal tissue alloantigen in mice of known H-2k and H-2a haplotypes. This antigen is not expressed in normal tissues of C3HfeB/HeN mice but is expressed in C3H/HeN mice, the strain from which the C3HfeB/HeN mice were originally derived. The present study indicates that spleen cells from C3HfeB/HeN and C3H/HeN mice respond reciprocally in the mixed lymphocyte reaction. Cytotoxic T lymphocytes specific for the tumor-associated alloantigen can be readily generated in mixed lymphocyte reactions in which spleen cells from C3HfeB/HeN mice are reacted with x-irradiated spleen cells from C3H/HeN or A strain mice. These cells are effective in suppressing the growth of the C3HfeB/HeN-derived lung tumor 85 in x-irradiated syngeneic recipients.     

The murine aromatic hydrocarbon responsiveness locus: a comparison of receptor levels and several inducible enzyme activities among recombinant inbred lines

The aromatic hydrocarbon responsiveness (Ah) locus has been correlated with genetic differences in the risk of drug toxicity, teratogenesis, chemical carcinogenesis, and mutagenesis. Hepatic cytosolic Ah receptor levels, 2-amino-5-chlorobenzoxazole (zoxazolamine) paralysis time following beta-naphthoflavone treatment and aryl hydrocarbon hydroxylase (AHH3, acetanilide 4-hydroxylase (Ac4H), and NAD(P)H:menadione oxidoreductase (NMOR)4, induction by 3-methylcholanthrene were studied in (a) the progenitors C57BL/6J (Ahb/Ahb) and DBA/2J (Ahd/Ahd) and 25 BXD recombinant inbred lines, (b) the progenitors C57BL/6N and C3H/HeN and 14 B6NXC3N recombinant inbred lines, and (c) the progenitors C57BL/6J and C3H/HeJ and 12 BXH recombinant inbred lines. The Ahb phenotype exhibits greater than 5 femtomole receptor/mg of cytosolic protein, less than or equal to 15 minutes zoxazolamine paralysis time, and twofold to 15-fold induction of these three hepatic enzyme activities; the Ahd phenotype exhibits less than or equal to 2 fmol receptor/mg protein, greater than 15 minutes zoxazolamine paralysis time, and less than 30% induction of these three activities. Among the BXD lines but especially among the B6NXC3N and BXH lines, high frequencies of recombination were found; the phenotype of each of the five parameters did not segregate with the phenotype of each of the other parameters in four or more recombinant lines. This report shows for the first time that AHH induction by 3-methylcholanthrene can occur in the Ahd phenotype mouse. These data underline the complexity of this genetic system when genes from C57BL/6 and DBA/2 are combined and particularly when genes from C57BL/6 and C3H/He inbred mouse strains are combined.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.     

Viability of frozen-thawed mouse embryos is affected by genotype

Embryos from mice of five different genotypes were evaluated for their ability to survive cryopreservation as measured by post-thaw in vitro development. In Study 1, ovulation was induced with a standardized pregnant mares' serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG) regimen, after which females were mated with males of the same genotype to produce incrossed embryos. Four- to 8-cell embryos were frozen in 1.5 M dimethyl sulfoxide (DMSO) at a rate of 0.5 degrees C/min to -80 degrees C and stored in liquid nitrogen. Following thawing at room temperature, embryos were cultured and development was evaluated 24 h later. The mean (+/- SEM) number of 4- to 8-cell embryos/pregnant female by stock/strain were: N:NIH(S), 6.8 +/- 0.8; N:NIH(S)-B, 5.8 +/- 0.5; N:GP(S), 6.5 +/- 0.6; C57BL/6N, 9.7 +/- 1.0; C3H/HeN MTV-, 9.5 +/- 0.9 (P less than 0.05). Post-thaw in vitro development was related to genetic background; the proportion of embryos culturing after thawing was: N:NIH(S), 49%; N:NIH(S)-B, 61%; N:GP(S), 66%; C57BL/6N, 75%; C3H/HeN MTV-, 56% (P less than 0.05). Study 2 was conducted to evaluate the influence of mating various females to males of a genotype known to have a lower post-thaw embryo survival rate. N:NIH(S)-B, N:GP(S), C57BL/6N, and C3H/HeN MTV- female mice were mated with N:NIH(S) males to produce hybrid embryos. Post-thaw embryo survival was reduced (P less than 0.05) in three of the four hybrid groups. Fresh incrossed and hybrid embryos from each study were cultured for 24 h and yielded culture rates ranging from 95% to 99% (P greater than 0.05) among all groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of intestinal goblet cells in the expulsion of Gymnophalloides seoi from mice

Role of intestinal goblet cells (GCs) in the expulsion of Gymnophalloides seoi was studied using 4 strains of mice, ICR, C3H/ HeN, BALB/c, and C57BL/6, after infection with 200 metacercariae isolated from oysters. On day 7 postinfection (PI), significantly higher (P < 0.05) worm recovery rates (WRRs) were observed in ICR (29.5 +/- 12.0%) and C3H/HeN (14.8 +/- 8.2%) than in BALB/c (5.7 +/- 5.3%) and C57BL/6 (0.8 +/- 1.1%) mice. Alteration of the GC mucins was marked in C57BL/6 mice. On day 14 PI, 5.2 +/- 5.2% and 0.6 +/- 0.7% of worms were recovered only from ICR and C3H/HeN mice. When C57BL/6 mice were immunosuppressed with prednisolone, WRR on day 7 PI increased to 11.7 +/- 13.9%, whereas the GC hyperplasia and mucin alteration diminished significantly. The results suggest that expulsion of G. seoi from the intestine is dependent on immune responses of the host, and GCs may be an important effector.     

Tumorigenicity of green turtle fibropapilloma-derived fibroblast lines in immunodeficient mice

Fibroblast lines derived from normal skin and spontaneous or experimentally induced fibropapillomas of green turtles (Chelonia mydas) were established and propagated in medium composed of a combination of Dulbecco's minimal essential with F12 medium plus 10% fetal bovine serum at 30 degrees C. Fibropapilloma-derived fibroblasts were indistinguishable from normal skin fibroblasts in vitro. Tumor lines did not exhibit loss of contact inhibition, anchorage independence, or reduced serum requirements. Inoculation of primary and early-passage tumor cells into the medial margin of the pinna of C57BL/6J-nu/nu, C.B17-scid/scid, or NOD-scid/scid mice, however, resulted in fibroma formation, whereas inoculation of normal skin fibroblasts did not. Tumor-derived cells inoculated into the flanks of mice did not form tumors. The turtle origin of fibroblasts in tumors from mouse ears was confirmed by immunohistochemical and karyotype analysis. Fibroblast lines that were established from mouse ear fibromas had the normal karyotype (modal 2N = 55) of C. mydas. The cooler anatomic sites (ears) of immunodeficient mice are useful for confirming the tumorigenic (transformed) phenotype of green turtle fibropapillomatosis-derived fibroblasts. This mouse ear tumorigenicity test should facilitate studies of mechanisms of cellular transformation in green turtle fibropapillomatosis and other neoplastic diseases of poikilothermic vertebrates.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Differences in antibody production against mouse hepatitis virus (MHV) among mouse strains

Several strains of mice were examined for antibody production after intranasal inoculation with a low virulence strain of mouse hepatitis virus (MHV), MHV-NuU. C57BL/6N mice were shown to be high responders in the production of complement fixing (CF) antibody as compared to C3H/HeN, BALB/c-AnN, DBA/2N mice. F1 hybrids B6C3 and BDF1 from C57BL/6N mice, showed CF antibody responses as high as C57BL/6N, suggesting that high responsiveness is genetically controlled. All these mouse strains were able to produce high titred neutralizing antibody to MHV.     

Refractoriness to migration inhibitory factor of macrophages of LPS nonresponder mouse strains.

The responsiveness to macrophage migration inhibitory factor (MIF) of peritoneal exudate cells (PEC) from the LPS unresponsive C3H/HeJ and C57BL/10ScCR mice was assessed by the indirect agarose microdroplet macrophage migration inhibition assay. No migration inhibition with PEC from C3H/HeJ nor C57BL/10ScCR mice was detected, whereas PEC from both C3H/HeN and C57BL/10Sn mice were significantly inhibited by even a 1/32 dilution of MIF-containing supernatants. Responsiveness to MIF of C3H/HeJ PEC could, however, be induced. In vivo inoculations of Mycobacterium bovis, strain BCG, 7 days before in vitro assay rendered C3H/HeJ PEC responsive to MIF. The lack of responsiveness to MIF by C3H/HeJ PEC appeared related to some form of suppression, since a mixture of PEC from C3H/HeN mice with 10 to 15% PEC from C3H/HeJ mice resulted in undetectable migration inhibition at any MIF dilution. In contrast to the usual lack of responsiveness of their macrophage to MIF, C3H/HeJ mice were able to produce MIK in response to PPD as well as their counterpart C3H/HeN mice after BCG sensitization. These results demonstrate that macrophages from C3H/HeJ and C57BL/10ScCR mice are unable to be inhibited in their in vitro migration of MIF (possibly being directly or indirectly influenced by a suppressor cell), whereas lymphoid cells from at least one of these strains, the C3H/HeJ mice, can produce MIF in response to antigenic stimulation.     

Interaction of thymus lymphocytes with histoincompatible cells. IV. Mixed lymphocyte reactions of activated thymus lymphocytes

CS7BL-activated CBA T cells (T.TDL) were obtained by thoracic duct cannulation of (CBA × C57BL)F₁ mice 4 days after heavy irradiation and injection of CBA thymus cells. T.TDL behaved differently from the TDL of normal CBA mice in unidirectional mixed lymphocyte culture in a number of respects: (a) the response of T.TDL was directed specifically against C57BL antigens, whereas normal TDL responded to both C57BL and BALB/c antigens; (b) the response of T.TDL was rapid but transient compared to that of TDL; (c) whereas only approximately 3% of TDL synthesized DNA specifically in response to C57BL antigens, as many as 25% of C57BL-activated T.TDL responded to these antigens. Evidence is presented which suggests that the T.TDL have a very limited capacity to proliferate. Most of the cells which responded to antigen synthesized DNA without subsequently entering mitosis.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods

We examined possible genotype effects on the survival of 8- to 16-cell mouse embryos isolated from four inbred strains (C57BL/6N, BALB/cAnN, DBA/2N, and C3H/HeN), a outbred stock (ICR), and various crosses after cryopreservation by vitrification or conventional slow freezing using glycerol solutions. The rates of in vitro development of C57BL/6N, BALB/cAnN, C3H/HeN, and ICR embryos to expanded blastocysts ranged from 86% to 94% after slow freezing and 85% to 97% after vitrification. The cryopreservation method did not significantly influence in vitro embryo survival after thawing (P >0.05). Although genotype significantly influenced the in vitro survival of embryos (P = 0.008), this presumably resulted from an increased difficulty in assessing the quality grade of C3H/HeN embryos prior to cryopreservation. The rates in vivo development of C57BL/6N, BALB/cAnN, C3H/HeN, DBA/2N, and ICR embryos to normal day 18–19 fetuses ranged from 19% to 64% after slow freezing and from 18% to 63% after vitrification. The in vivo development of cryopreserved embryos was significantly influenced by cryopreservation method and genotype (P = 0.01 and P = 0.001, respectively). Vitrification yielded significantly higher rates of in vivo development than that after slow freezing (P > 0.05). In vivo development rates of DBA/2N and ICR♀ X B6D2F1 ♂ embryos after cryopreservation were significantly higher than that of embryos from BALB/cAnN and C3H/HeN mice (P < 0.05). These results indicate that parental genotype exerts little or no effect on the ability of embryos to develop in vitro after vitrification or slow freezing. Differences in the ability of cryopreserved embryos to develop normally in vivo may reflect inherent genotype related differences in their post-implantation developmental potential and not their sensitivity to cryoinjury. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.