Marker tolerant,immunocompetent animals as a new tool for regenerative medicine and long-term cell tracking

Background  

Immune-mediated rejection of labeled cells is a general problem in transplantation studies using cells labeled with any immunogenic marker, and also in gene therapy protocols. The aim of this study was to establish a syngeneic model for long-term histological cell tracking in the absence of immune-mediated rejection of labeled cells in immunocompetent animals. We used inbred transgenic Fischer 344 rats expressing human placental alkaline phosphatase (hPLAP) under the control of the ubiquitous R26 promoter for this study. hPLAP is an excellent marker enzyme, providing superb histological detection quality in paraffin and plastic sections.     

Hematopoietic bone marrow cells participate in endothelial, but not epithelial or mesenchymal cell renewal in adult rats

The extent to which bone marrow (BM) contributes to physiological cell renewal is still controversial. Using the marker human placental alkaline phosphatase (ALPP) which can readily be detected in paraffin and plastic sections by histochemistry or immunohistochemistry, and in ultrathin sections by electron microscopy after pre-embedding staining, we examined the role of endogenous BM in physiological cell renewal by analysing tissues from lethally irradiated wild-type inbred Fischer 344 (F344) rats transplanted (BMT) with unfractionated BM from ALPP-transgenic F344 rats ubiquitously expressing the marker. Histochemical, immunohistochemical and immunoelectron microscopic analysis showed that the proportion of ALPP(+) capillary endothelial cells (EC) profoundly increased from 1 until 6 months after BMT in all organs except brain and adrenal medulla. In contrast, pericytes and EC in large blood vessels were ALPP(-) . Epithelial cells in kidney, liver, pancreas, intestine and brain were recipient-derived at all time-points. Similarly, osteoblasts, chondrocytes, striated muscle and smooth muscle cells were exclusively of recipient origin. The lack of mesenchymal BM-derived cells in peripheral tissues prompted us to examine whether BMT resulted in engraftment of mesenchymal precursors. Four weeks after BMT, all haematopoietic BM cells were of donor origin by flow cytometric analysis, whereas isolation of BM mesenchymal stem cells (MSC) failed to show engraftment of donor MSC. In conclusion, our data show that BM is an important source of physiological renewal of EC in adult rats, but raise doubt whether reconstituted irradiated rats are an apt model for BM-derived regeneration of mesenchymal cells in peripheral tissues.     

A simple in vivo approach to investigate invasive trophoblast cells

Intrauterine trophoblast cell invasion is an essential part of hemochorial placentation. Aberrant trophoblast cell invasion has been associated with pathologies including preeclampsia and fetal growth restriction. In this study, we describe an in vivo method to assess trophoblast cell invasion using a transgenic rat model, constitutively expressing heat stable human placental alkaline phosphatase (Rosa 26 promoter driven human placental alkaline phosphatase, R26-hAP). Wild-type female Fischer 344 inbred rats were mated with hemizygous R26-hAP transgenic male Fischer 344 rats and sacrificed during the second half of pregnancy. Heat stable alkaline phosphatase (AP) activity associated with the invasive transgenic trophoblast cells was monitored in the wild-type uterine mesometrial compartment and used as an index of trophoblast cell invasion. The expression pattern of cytokeratins by invasive trophoblast cells mimicked the uterine mesometrial distribution of AP activity. Trophoblast cell invasion exhibited a gestation-dependent profile with peak invasion between days 18-20 of pregnancy. In summary, we have devised a simple in vivo method for assessing intrauterine trophoblast cell invasion. This technique should facilitate the discovery of endogenous regulatory mechanisms controlling trophoblast cell invasion and should represent an effective method of testing the impact of various environmental stressors on an essential part of hemochorial placentation.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

Derivation of a Germline Competent Transgenic Fischer 344 Embryonic Stem Cell Line

Embryonic stem (ES) cell-based gene manipulation is an effective method for the generation of mutant animal models in mice and rats. Availability of germline-competent ES cell lines from inbred rat strains would allow for creation of new genetically modified models in the desired genetic background. Fischer344 (F344) males carrying an enhanced green fluorescence protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. After establishment of ES cell lines, rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing was conducted in selected ES cell lines. One male ES cell line, F344-Tg.EC4011, was further evaluated for germline competence by injection into Dark Agouti (DA) X Sprague Dawley (SD) blastocysts. Resulting chimeric animals were bred with wild-type SD mates and germline transmissibility of the ES cell line was confirmed by identification of pups carrying the ES cell line-derived EGFP transgene. This is the first report of a germline competent F344 ES cell line. The availability of a new germline competent ES cell line with a stable fluorescence reporter from an inbred transgenic rat strain provides an important new resource for genetic manipulations to create new rat models.     

Comparison of rat mandible bone characteristics in f344 substrains, f344/du and f344/n.

The characteristics of the mandible bone were compared through DXA methods between two major substrains of F344 rats, F344/DuCrlCrlj and F344/NSlc at around 60 days of age. Since these two substrains are clearly different in survival and mandible morphology, some genetic differences are supposed to exist. In contrast to a previous microsatellite analysis, clear and significant differences were detected in the body and mandible weights, the mandible bone mineral contents (BMC), bone area (AREA), bone mineral density (BMD) and bone mineral ratio (BMR), between F344/DuCrlCrlj and F344/NSlc, with the mandible molar teeth intact in the bone. Thus, care is needed in the experimental use of these substrains, as results may differ between them. The newly proposed parameter, BMR, may especially contribute to the comparison of bone characteristics among species.     

Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes

To understand the differences between the rat intestinal alkaline phosphatase isozymes rIAP-I and rIAP-II, we constructed structural models based on the previously determined crystal structure for human placental alkaline phosphatase (hPLAP). Our models of rIAP-I and rIAP-II displayed a typical alpha/beta topology, but the crown domain of rIAP-I contained an additional beta-sheet, while the embracing arm region of rIAP-II lacked the alpha-helix, when each model was compared to hPLAP. The representations of surface potential in the rIAPs were predominantly positive at the base of the active site. The coordinated metal at the active site was predicted to be a zinc triad in rIAP-I, whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP-II. Using metal-depleted extracts from rat duodenum or jejunum and hPLAP, we performed enzyme assays under restricted metal conditions. With the duodenal and jejunal extract, but not with hPLAP, enzyme activity was restored by the addition of zinc, whereas in nonchelated extracts, the addition of zinc inhibited duodenal IAP and hPLAP, but not jejunal IAP. Western blotting revealed that nearly all of the rIAP in the jejunum extracts was rIAP-I, whereas in duodenum the percentage of rIAP-I (55%) correlated with the degree of AP activation (60% relative to that seen with jejunal extracts). These data are consistent with the presence of a triad of zinc atoms at the active site of rIAP-I, but not rIAP-II or hPLAP. Although no differences in amino acid alignment in the vicinity of metal-binding site 3 were predicted between the rIAPs and hPLAP, the His153 residue of both rIAPs was closer to the metal position than that in hPLAP. Between the rIAPs, a difference was observed at amino acid position 317 that is indirectly related to the coordination of the metal at metal-binding site 3 and water molecules. These findings suggest that the side-chain position of His153, and the alignment of Q317, might be the major determinants for activation of the zinc triad in rIAP-I.     

Slc:Wistar outbred rats show close genetic similarity with F344 inbred rats

Although Slc:Wistar rats are used widely in biomedical research as outbred rats, close similarities in growth curves, survival rates, and immunological and biochemical phenotypes have been reported between Slc:Wistar and F344 inbred rats. We reported previously that nine genetic variations that were fixed in Slc:Wistar rats had identical genotypes in F344 rats. Here, we examined the genetic characteristics of Slc:Wistar rats using 27 simple-sequence length polymorphism (SSLP) markers and compared them with other Wistar stocks available in Japan and with some F344 strains. Among 27 SSLP loci, 23 (85%) were fixed in the Slc:Wistar rats, which was the highest among the other Wistar stocks. The 23 fixed loci shared identical genotypes with corresponding loci in F344 rats. Further, the predominant allele types in the unfixed loci had allele frequencies as high as 80%, and these alleles were identical in the F344 rats. When the nine genetic variations reported previously are added, a total of 32 (89%) out of the 36 loci examined were fixed and identical in the Slc:Wistar and F344 rat genomes. These findings indicate the low genetic variation in Slc:Wistar rats and the high genetic similarity between the Slc:Wistar and F344 inbred rats. This study demonstrates the importance of characterizing outbred rats and the need to pay ample attention to the genetic characteristics the Slc:Wistar rats for their proper use.     

Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication we describe our studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 rats reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation.     

Lectin--digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry.

We report the use of a novel hapten system for lectin cytochemistry. Various lectins conjugated to the steroid hapten digoxigenin (DIG) and monospecific anti-digoxigenin antibodies were applied for the light and electron microscopic detection of glycoconjugates in tissue sections. Both IgG and Fab' anti-DIG antibodies were complexed to particles of colloidal gold and compared to commercially available alkaline phosphatase and horseradish peroxidase conjugated Fab' as general second step reagents. The three different markers performed equally well on paraffin sections whereas the gold-labeled antibodies were superior reagents for semithin and ultrathin sections of Lowicryl K4M embedded tissues. In conjunction with the latter marker, no pretreatment to abolish endogenous enzyme activity was necessary. At the light microscope level, gold signal amplification by the photochemical silver reaction was required. DIG, in contrast to biotin, does not occur in animal tissues thus eliminating the need for blocking reactions prior to lectin incubation. Compared to affinity techniques using glycoprotein-gold complexes as second step reagent the DIG hapten system required smaller amounts of lectins. The staining patterns were indistinguishable from those obtained in other lectin-gold techniques and the specificity of the labeling could be demonstrated in sugar inhibition tests.     

Lectin — digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry

Summary We report the use of a novel hapten system for lectin cytochemistry. Various lectins conjugated to the steroid hapten digoxigenin (DIG) and monospecific anti-digoxigenin antibodies were applied for the light and electron microscopic detection of glycoconjugates in tissue sections. Both IgG and Fab' anti-DIG antibodies were complexed to particles of colloidal gold and compared to commercially available alkaline phosphatase and horseradish peroxidase conjugated Fab' as general second step reagents. The three different markers performed equally well on paraffin sections whereas the gold-labeled antibodies were superior reagents for semithin and ultrathin sections of Lowicryl K4M embedded tissues. In conjunction with the latter marker, no pretreatment to abolish endogenous enzyme activity was necessary. At the light microscope level, gold signal amplification by the photochemical silver reaction was required. DIG, in contrast to biotin, does not occur in animal tissues thus eliminating the need for blocking reactions prior to lectin incubation. Compared to affinity techniques using glycoprotein-gold complexes as second step reagent the DIG hapten system required smaller amounts of lectins. The staining patterns were indistinguishable from those obtained in other lectin-gold techniques and the specificity of the labeling could be demonstrated in sugar inhibition tests.     

Effect of melatonin on bone metabolism in ovariectomized rats.

To assess the effect of pharmacological dose of melatonin on bone metabolism in ovariectomized rats, urinary deoxypyridinoline (a marker of bone resorption) and calcium excretion, circulating levels of calcium, phosphorus and bone alkaline phosphatase activity (a marker of bone formation), and bone mineral density (BMD), mineral content (BMC) and bone area (BA) of total body, were measured in adult rats for up to 60 days after surgery. Rats received melatonin in the drinking water (25 microg/ml water) or drinking water alone. Urinary deoxypyridinoline increased significantly after ovariectomy by 51% (30 days after surgery) and by 47% (60 days after surgery). The increase in urinary deoxypyridinoline found 30 days after ovariectomy was not observed in melatonin-treated rats. Urinary calcium concentration was similar in the 4 experimental groups studied, as was the circulating calcium concentration at every time interval examined. Fifteen days after surgery, a significant increase in serum phosphorus and bone alkaline phosphatase levels occurred in ovariectomized rats receiving melatonin as compared to their controls. Sixty days after surgery BMD, BMC and BA decreased significantly in ovariectomized rats, an effect not modified by melatonin. Serum estradiol decreased significantly by 30 days after ovariectomy to attain values close to the limit of detection of the assay by 60 days after ovariectomy. The results support the conclusion that a pharmacological amount of melatonin modifies bone remodeling after ovariectomy and that the effect may need adequate concentrations of estradiol.     

Immunocytological investigation of protein synthesis in Escherichia coli.

Ferritin-conjugated specific antibodies have been used to localize beta-galactosidase and both the monomer and active dimer of alkaline phosphatase in frozen thin sections of cells of Escherichia coli O8 strain F515. The even distribution of the ferritin marker throughout cells that had been induced for beta-galactosidase synthesis, frozen, sectioned, and exposed to ferritin-anti-beta-galactosidase conjugate showed that this enzyme was present throughout the cytoplasm of these cells. Frozen thin sections of cells that had been derepressed for the synthesis of alkaline phosphatase were exposed to both ferritin-anti-alkaline phosphatase monomer and ferritin-anti-alkaline phosphatase dimer conjugates, and the ferritin markers showed a peripheral distribution of both the monomer and the dimer of this enzyme. This indicates that alkaline phosphatase is present only in the peripheral regions of the cell and argues against the existence of a cytoplasmic pool of inactive monomers of this enzyme. This peripheral location of both the monomers and dimers of alkaline phosphatase supports the developing concensus that this enzyme is, like other wall-associated enzymes, synthesized in association with the cytoplasmic membrane and vectorially transported to the periplasmic area, where it assumes its tertiary and quaternary structure and acquires its enzymatic activity.     

Orchiectomized Fischer 344 male rat models body composition in hypogonadal state

The hypogonadal state in men is accompanied by substantial decreases in muscle and bone mass and by an increase in adiposity. Most of the strains of orchiectomized (ORX) rat that have been used to model this state display substantial losses in bone, but only subtle changes in adiposity and muscle mass. In order to identify a rat model displaying a robust catabolic response to ORX, we studied three strains: Fischer 344 (F344), Brown Norway and Wistar. ORX caused a significant and sustained decrease in weight gained by F344, but only a trend toward reduced weight gain in Brown Norway rats and a modest reduction weight gain in Wistar rats that was significant only after 56days. ORX suppressed food intake in F344 rats, and to a lesser degree in Brown Norway and Wistar rats. ORX reduced muscle mass significantly in F344 rats, but not in Brown Norway or Wistar rats. ORX increased adiposity moderately in F344 rats and substantially in Wistar rats. ORX caused a marked reduction in prostate mass and increase in bone resorption in all three strains. Thus, F344 was the only strain in which ORX produced substantial decreases in food intake, body weight and muscle mass with increased adiposity and increased bone resorption. We conclude that the F344 rat displays a broad range of catabolic effects following ORX and is the best rat model for studying the androgenic pathway and strategies for reversing catabolic changes induced by hypogonadism.     

骨髓标本中免疫组织化学及DNA原位末端标记双重染色技术的应用

为扩展免疫酶标记分子病理学技术在血液病基础与临床研究中的应用,我们探索了碱性磷酸酶抗碱性磷酸酶（A-PAAP）免疫组化和DNA原位末端标记（ISEL）技术在骨髓涂片和切片（乙二醇甲基丙烯酸酯GMA冷包埋）中联合应用（双重染色）的方法,结果令人满意,已初步用于白血病分化抗原及细胞凋亡的共同检测。     

The beneficial effect of OST-6 (OsteoCare), a herbomineral formulation, in experimental osteoporosis.

OST-6 (OsteoCare), a herbomineral formulation, was evaluated for its inhibitory effect on the progress of bone loss induced by ovariectomy in rats. Ovariectomized (Ovx) rats were administered with OST-6 at 250 and 500 mg/kg b.wt., orally daily for 90 days. On 91st day, ovariectomized rats showed reduced bone mineral content and increased serum alkaline phosphatase levels, excretion of urinary calcium and pyridinium cross-links levels. Histologically, bone sections revealed narrowed and disappearance of trabeculae and widened medullary spaces. The total numbers of Tartrate-resistant acid phosphatase (TRAP) positive cells were significantly increased both in-vivo and in-vitro methods. OST-6, at a dose of 500 mg/kg, significantly improved bone mineral contents, serum alkaline phosphatase levels, reduced the elevated urinary calcium and pyridinium cross-links excretion, number of TRAP positive cells and reversal of the above mentioned histological features. These results indicate the usefulness of OST-6 in the management of osteoporosis in a natural way through herbal resources.     

CD26 expression determines lung metastasis in mutant F344 rats: involvement of NK cell function and soluble CD26

The association between CD26 expression, tumor cell adhesion, metastasis, and natural killer (NK) cell function was investigated in a CD26 mutant Fischer 344 (F344/DuCrj) substrain from Japanese breeders (F344JAP) in comparison with wild-type F344 substrains from US (F344/Crl) and Hannover (HAN; F344/Ztm) breeders. F344JAP rats lack the dipeptidyl peptidase IV activity of CD26 and show a reduced cell surface expression of the mutated CD26 glycoprotein. In vivo adhesion of vital dye-labeled MADB106 tumor cells, tumor colonization, CD26 enzymatic activity, and CD26 immunoreactivity in lungs and soluble CD26-like protein expression in serum were markedly reduced in F344JAP rats. These findings demonstrate that CD26 protein expression exerts a key role in lung metastasis. In addition, NK cell cytotoxicity against MADB106 cells was diminished in the mutant F344 substrain, suggesting that CD26 enzymatic activity sustains NK cytotoxicity. Interestingly, tumor cells lacked CD26 immunoreactivity in vitro, but displayed CD26 immunoreactivity in situ after in vivo inoculation as well as after incubation with rat serum, indicating that soluble CD26-like protein assembles in tumor cells during in vivo passage, which may interact with the process of tumor adhesion and metastasis. Overall, these findings indicate that altered expression and function of a single enzyme-the CD26 protein--can drastically change the outcome of metastatic disease.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.     

Improvements in histological quality and signal retention following in situ hybridization in early chick embryos using plastic resin and recolorization.

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.