Histochemical characteristics of glycoproteins in the bile duct system of mice immunized with swine serum

Summary The bile duct system of BALB/c and DDY mice, which were immunized with swine serum (SS) or not, was examined histochemically. Biliary epithelial cells of the SS-treated BALB/c mice, which were positively stained with periodic acid-Schiff (PAS) and had binding sites of Dolichos biflorus (DBA), were thought to secrete neutral glycoproteins with terminal N-acetyl-d-galactosamine residues. Those of the SS-treated DDY mice were however negatively or weakly stained with any histochemical stainings. On the other hand, glandular epithelial cells of the SS-treated mice of both strains, which were positively stained with high iron diamine-alcian blue (HID-AB) and had binding sites of DBA, Griffonia simplicifolia-II (GS-II), Ulex europaeus-I (UEA-I), and Triticum vulgaris (WGA), were thought to secrete glycoproteins with terminal sialic acid residues. Biliary and glandular epithelial cells of the normal mice contained only a small amount of glycoproteins showing similar histochemical characteristics to those in the SS-treated BALB/c mice. BALB/c mice immunized with SS were thought to be very useful for the investigation of production and secretion of glycoproteins in the bile duct system as well as being good model of bile duct disease.     

Mouse strain difference in bile duct lesions induced by swine serum injections

The mouse strain difference in bile duct lesions was studied on male A/J, BALB/c, C57BL/6, C3H/He, DBA/2 and DDY mice 4 weeks old given intraperitoneal injections of swine serum (0.05 or 0.2 ml per mouse) twice a week for 4 weeks. The hepatic lesions were restricted to the portal tract. Biliary epithelial cells showed hypertrophy and hyperplasia, and eosinophilic and homogeneous or needle-shaped material appeared in the cytoplasm of such hypertrophied epithelial cells and in the ductular lumen. Around these damaged biliary epithelia, eosinophil leukocyte and plasma cell infiltration with proliferation of collagen fibres was commonly detected. These changes became more apparent with increasing size of bile duct. Such histopathological characteristics of hepatic lesions were essentially the same in all strains, but the severity showed a clear strain difference: the lesion was marked in the DDY, A/J and BALB/c strains, moderate in C3H/He and slight in C57BL/6 and DBA/2. A high production of anti-swine-serum antibodies associated with a marked increase in the number of mouse IgG-producing lymphocytes in the spleen was detected in the strains showing the marked hepatic lesions.     

Histochemical study on the bile duct system of normal Mongolian gerbils

The bile duct system of normal Mongolian gerbils was examined histochemically. The luminal surface membrane and apical cytoplasm of the biliary and gallbladder epithelial cells were stained with periodic acid-Schiff (PAS), alcian blue, pH 2.5 (AB) and high iron diamine (HID)-AB, and many epithelial cells of the common bile duct and gallbladder had weakly PAS-positive granular material in their supranuclear cytoplasm. Lectin-histochemically, these cells had binding sites to Concanavalia ensiformis (ConA), Dolichos biflorus (DBA), Glycine max (SBA), Ulex europeas-I (UEA-I), and Triticum vulgaris (WGA). On the other hand, the periductal glandular epithelial cells were not stained by any histochemical stainings. In addition to these light microscopic findings, the electron microscopic findings based on the periodic acid-silver methenamine method and avidin-biotin colloidal gold method for DBA and WGA suggested that the biliary and gallbladder epithelial cells of Mongolian gerbils secreted mucin with terminal sialic and sulfonic acid residues and that the lectin binding activity of mucin secreted from these cells was similar to that of mucin secreted from the periductal glandular epithelial cells of mice and rats.     

Effect of active immunization against a recombinant mouse granulocyte-macrophage colony-stimulating factor/somatostatin fusion protein on the growth of mice

Female BALB/c mice were actively immunized subcutaneously with a recombinant protein of granulocyte-macrophage colony-stimulating factor (GM-CSF) fused with somatostatin (SS) (GM-CSF/SS). Fifty-four days after the primary immunization, the body weight of the immunized mice increased by 4.62% compared with the control (P < 0.05), together with the induction of detectable serum antibodies against SS. The level of serum growth hormone (GH) elevated by 44.54% (P < 0.05) and the mRNA expression of muscular IGF-1 increased by 94% for the GM-CSF/SS-treated mice. The results indicated that the recombinant protein GM-CSF/SS was efficient in inducing specific immunity against SS, subsequently leading to the increase of the GH level by SS neutralization, and ultimately improving the growth of mice.     

Foreign serum-induced bile duct lesion (BDL) in athymic BALB/c nude mice

To investigate a role of cellular immunity in foreign serum-induced bile duct lesion (BDL) in mice, athymic BALB/c nude (nu/nu) mice were intraperitoneally injected with swine serum (SS) twice a week up to 8 weeks and were compared with euthymic BALB/c heterozygote (nu/+) and wild-type (+/+) mice treated with SS in the same way for 4 weeks. All immunized nu/+ and +/+ mice developed marked BDL, and their sera showed high anti-SS IgE and IgG1 antibody titers, whereas no immunized nu/nu mice developed lesions, and their sera showed no elevation of antibody titers. Next, nu/nu mice were reconstituted with splenocytes derived from nu/+ mice, and then were intraperitoneally injected with SS twice a week for 3 weeks. Most of the reconstituted nu/nu mice developed BDL, and their sera showed the elevation of anti-SS IgE and IgG antibody titers. These results suggest that cellular immunity may play a pivotal role in the pathogenesis of swine serum-induced BDL.     

Histochemical study on the bile duct system of normal rats

The bile duct system of normal rats was examined histochemically. Although the apical surface of biliary and glandular epithelial cells was positively stained with Concanavalia ensiformis (Con A), Dolichos biflorus (DBA), Glycine max (SBA), Ulex europeus-I (UEA-I) and Triticumvulgaris (WGA), the cytoplasm did not apparently react with any lectins. Therefore these cells might not play an important role in an active secretion of mucin. On the other hand, the cytoplasm of goblet cells was positively stained with periodic acid-Schiff, high iron diamine, Con A, DBA, Griffonia simplicifolia-II (GS-II), SBA, UEA-I, and WGA. This suggests that mucin secreted from the goblet cells may be acid one with sulfonic terminals.     

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. II. Rat

Summary Salivary glands and pancreases from male rats were stained with a battery of ten different lectin-horseradish peroxidase conjugates. Qualitative and quantitative differences were observed in the content of terminal sugar residues in stored secretory glycoproteins in parenchymal cells of glands having a similar histological structure. Heterogeneity in the content of secretory glycoconjugates was also found between cells in the same exocrine glands, which were previously thought to be identical on the basis of classical morphological and histochemical staining studies. Similar differences were observed in the structure of glycoconjugates associated with the apical surface of epithelial cells lining glandular excretory ducts. Intercalated ducts presented a gland specific staining pattern different from that of the glandular secretory cell population, whereas striated duct and interlobular duct epithelial cells stained similarly in all major rat exocrine glands. A comparison of lectin binding patterns in identical histological sites in the mouse, reported in a companion paper, is provided, and the similarities and differences between these two rodent species are discussed. In addition to providing valuable information concerning the localization and structure of tissue complex carbohydrates, a comparison of staining in the same tissue sites with labelled lectins reported biochemically to have similar binding specificity has revealed interesting differences in the binding specificity of these macromolecules.     

Spontaneous heart failure in BALB/c mice

The hearts of BALB/c mice are known to acquire pronounced greyish white spots (cardiac white spots). BALB/c male mice were examined for the relationship between the incidence of cardiac white spots and weekly age, and compared with DDY male mice. During the observation period of 0.4-30 weeks, cardiac white spots on the right ventricle of BALB/c mice were first detected at three weeks (6 of 20 mice; 30%), and the maximal incidence of cardiac white spots was obtained at nine weeks (39 of 44 mice; 88%). In contrast, DDY mice were completely devoid of cardiac spots. Histopathologically, the cardiac spots were dystrophic calcinosis. There were significant increases in the relative organ weights of the heart and kidney of BALB/c mice compared with those of DDY mice. However, there was no significant difference between BALB/c and DDY mice in serum calcium concentration or histological characteristics of the parathyroid gland or bone marrow. The cardiac white spots of BALB/c mice were considered to be controlled by genetic susceptibility that occurred spontaneously with aging. The results described here suggest that BALB/c mice are adequate experimental animals for the study of myocardial disease that occurs spontaneously.     

Histochemical study of rabbit duodenal mucosa carried out using lectins

The Authors report histochemical findings about rabbit's duodenal mucosa. The present study has been carried out using five different lectins (Peanut Agglutinin (PNA), Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), Soybean Agglutinin (SBA), Ulex Europaeus Agglutinin I (UEA-I). These lectins have been labelled with Horseradish Peroxidase and binding sites have been stained with 3-3' Diaminobenzidine, according to Farragiana et al. The PNA reacted with the glandular cells, while the reaction was negative in the superficial cells. The DBA reacted exclusively with the glandular cells. The superficial and the glandular cells showed strong positive binding sites to the WGA and slight positive binding sites to the SBA. The UEA-I did not react with the epithelial cells. The presence of binding sites for the lectins we have used in the present study, shows a different glycoprotein composition of the cellular secretion, in comparison with the other animals we have already studied. In addition, these lectins can not be used as cellular differentiation markers in the epithelial cells of the rabbit's duodenal mucosa.     

Hepatic homing of mononuclear inflammatory cells isolated during murine chronic graft-vs-host disease

Liver injury in murine chronic graft-vs-host disease (CGVHD) to minor histocompatibility Ag, B10.D2----BALB/c (600 rad), is characterized by mononuclear cell inflammation and necrosis of interlobular bile ducts. Bile duct destruction in this model is similar to that which occurs in human CGVHD, late liver transplant rejection, and primary biliary cirrhosis. This model provides a unique opportunity to isolate mononuclear inflammatory cells from the liver during CGVHD, study their functions, and investigate the immunologic mechanisms responsible for bile duct destruction. In the present study, we compared the in vivo organ homing of mononuclear inflammatory cells (MC) isolated from the liver and spleen during the course of CGVHD. MC isolated from the liver showed a progressive increase in homing to the livers of BALB/c mice from day 7 through 42. In contrast, the hepatic homing of MC isolated from the spleen peaked at day 21 and subsequently declined. CGVHD spleen MC showed a progressive increase in homing to the spleen of BALB/c mice whereas CGVHD liver MC showed no change over time. Homing to other organs was negligible. The hepatic and splenic homing of MC isolated during CGVHD was significantly greater in BALB/c (host) mice than in B10.D2 (donor) mice. Autoradiography was used to determine the intrahepatic sites at which CGVHD liver MC accumulate after i.v. injection into BALB/c mice. The results indicated that MC isolated from the liver when bile duct inflammation is most intense accumulate preferentially in hepatic portal spaces in close proximity to interlobular bile ducts. These results suggest that hepatic homing by CGVHD liver MC is specific for minor histocompatibility Ag expressed on host biliary epithelial cells. These data support the hypothesis that bile duct destruction in murine CGVHD is mediated by MC that are sensitized to minor histocompatibility Ag expressed by host biliary epithelial cells.     

Egg production of Clonorchis sinensis in different strains of inbred mice.

In order to compare the intraspecific variation in host-parasite relationship of Clonorchis sinensis, six strains of inbred mice, ICR, DDY, GPC, BALB/c, nude and DS, were infected orally with 20 metacercariae of C. sinensis. The biologic incubation period of C. sinensis was the shortest in DDY mice, 21.2 days in average, followed by GPC 21.4, BALB/c and DS 23.2, ICR and nude 23.4 days, respectively. The fertile period of the fluke was also the longest in the DDY strain, 164 days on average, followed by GPC 132, BALB/c 97, nude 37, DS 32 and ICR 28 days. The egg-laying capacity of the fluke in DDY and GPC was relatively high and stable compared with the other four strains of mice. It was found that there are intraspecific variations in biologic incubation period, fertile period, and fecundity of C. sinensis. The DDY mouse is likely to be the most suitable experimental animal among the six strains of the mice tested.     

Impulsive choice in inbred strains of mice

When humans or non-humans are given a choice between receiving a sooner-smaller (SS) reinforcer, or a later-larger (LL) reinforcer, the choice of a SS reinforcer represents impulsive choice whereas the choice of a LL reinforcer represents self-controlled choice. It has been suggested that both biological and genetic factors influence impulsive/self-control choice in this paradigm. In the present study, the inbred strains of BALB/c, C57BL/6, and DBA/2 mice were given a choice to press one of two levers in an operant chamber. Depending on their choice, mice received either a smaller reinforcer (one pellet delivered after 6s) sooner (SS) or a larger reinforcer (two pellets after 6, 9, 12, 18, or 30s) later (LL). Mice preference for the larger reinforcer decreased with longer delays. More importantly, the BALB/c mice chose the SS reinforcer more often than the C57BL/6 mice under the 9- and 12-s delays, and more often than the DBA/2 mice under the 9-s delay. This indicates that the choice pattern of the BALB/c strain is more "impulsive" than the other strains and suggests that specific gene configurations influence impulsive choice in mice.     

Ethanol-induced hypothermia in mice: Influence of genotype on development of tolerance

Male mice of BALB/c, C57BL, DBA/2 strains and two lines of mice selectively bred for sensitivity to ethanol, Long-Sleep (LS) and Short-Sleep (SS), were tested for ethanol-induced hypothermia following varied doses of ethanol. The results show that the genotype as well as the dose of the drug determines the intensity and the duration of the effect. Repeated injection of ethanol results in the decrease of hypothermia in BALB/c mice and in C57BL, but not in DBA/2 mice, indicating that tolerance as measured in this study may not develop in certain genotypes of mice. The blood ethanol elimination data after repeated injections of ethanol indicate that the metabolic factors do not explain the changes observed in the hypothermic effects of repeated injections of ethanol.     

Immunodepressant action of cyclophosphamide in different strains of mice

A study was made of the immunodepressive effect of cyclophosphamide (CP) on mice of 3 strains (BALB/c, CBA, and DBA/2) immunized with sheep red blood cells (SRBC). With the optimal immunizing dose of the antigen (5 X 10(8) SRBC) the most pronounced immunodepression was noted in DBA/2 mice, and with the high dose (6.2 X 10(9))--in DBA/2 and CBA mice. The CP action proved to depend on the dose of the antigen administered; in BALB/c mice a reduction in the number of the antibody-forming cells was the same with both SRBC doses, in DBA/2 mice an increase of the antigen dose led to reduction of immunode pression, and in CBA mice -- to its enhancement (with sufficiently high CP doses). Determination of the rate of oxidative CP hydroxylation by the liver microsomes of mice showed it to be comparatively low in DBA/2 and CBA mice, and much greater in BALB/c mice. It is supposed that the detected differences in the immunodepressive action of CP could be connected with different sensitivity of the target cells and (or) with the peculiarities of its metabolism in mice belonging to different strains.     

Detection of micronucleated cells and gene expression changes in glandular stomach of mice treated with stomach-targeted carcinogens

To assess the genotoxicity of chemicals on the stomach, we developed in vivo assays that can detect micronucleus induction and gene expression changes in epithelial cells of the glandular stomach in mice. Male BALB/c mice were orally given a single dose (100 mg/kg) of N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as stomach-targeted carcinogens. The glandular stomach was excised at 4h, 3 and 4 days after administration, and a single cell suspension of epithelial cells was prepared from the everted glandular stomach by EDTA treatment. For determination of micronucleus induction, gastric epithelial cells on days 3 and 4 after administration were fixed with 10% neutral-buffered formalin, stained with a combination of AO-DAPI, and analyzed under fluorescence microscopy. We also examined the induction of micronuclei in peripheral blood of these mice on days 2 and 3 after administration. Moreover, total RNA was extracted from gastric epithelial cells at 4h after administration, and p21 and plk2 expression was analyzed using a quantitative RT-PCR technique. 1) A significant increase of micronucleated cells was observed in the glandular stomach in mice treated with N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to mice treated with vehicle. 2) In peripheral blood, induction of micronuclei was observed in mice treated with MNU but not with MNNG. 3) p21 and plk2, which related to cell cycle arrest, were up-regulated in the glandular stomach in mice treated with MNU or MNNG compared to mice treated with vehicle. The present study showed that these assays using glandular stomach may help to evaluate the genotoxicity of chemicals after oral administration.     

Histochemical localization of estrogen induced sulfated glycoprotein in rabbit uterus

Summary Histochemical localization of the estrogen-induced sulfated glycoproteins was made in the estrogen-treated rabbit uterus. Biochemical studies by a group of Endoet al. affirmed these particular glycoproteins were PAS-positive and metachromatic as stained with TB. No sign of digestion, however, has been detected in a series of tests with α-amylase, testicular hyaluronidase, streptomyces hyaluronidase, chondroitinase AC and chondroitinase ABC, and heparinase. The apical portions of the epithelial and glandular cells, obviously expanded by the estrogen treatment, display strong β-metachromasia with TB (pH 4.0), saliva-resistant PAS-positive reactions, and also alcianophilia with AB (pH 2.5). These reactions are not reduced after the treatment with the enzymes above-mentioned. Meanwhile, in the stromal matrix, the same enzymes give an influence to diminish the reactions to various extent. Our results suggest that the estrogen-induced sulfated glycoprotein is definitely localized in the apical portions of the epithelial and glandular cells. The identity is emphasized between the substance that is elucidated in the histochemical sections and the sulfated glycoproteins that have been specified solely by means of biochemical assays. This work has been supported by grants from the Population Council, New York.     

A novel method for establishment and characterization of extrahepatic bile duct epithelial cells from mice

Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic bile duct epithelial cell culture. Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts’ epithelial cells and cultured on rat tail type I collagen-coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10 ng/ml epidermal growth factor at 37°C in an incubator with 5% humidified CO₂. The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within 5–6 d after culture; they were positive against anticytokeratin-19 immunostaining. Transmission electron microscopy showed typical bile duct epithelia with microvilli on the cytomembrance, Golgi complex, massive mitochondria, and rough endoplasmic reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal sigmoidal growth curve. This culture technique might be a reliable method for isolation, purification, and primary culture of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the pathophysiology of hepatobiliary diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases.     

Histochemical localization of estrogen induced sulfated glycoprotein in rabbit uterus.

Histochemical localization of the estrogen-induced sulfated glycoproteins was made in the estrogen-treated rabbit uterus. Biochemical studies by a group of Endo et al, affirmed these particular glycoproteins were PAS-positive and metachromatic as stained with TB. No sign of digestion, however, has been detected in a series of tests with alpha-amylase, testicular hyaluronidase, streptomyces hyaluronidase, chondroitinase AC and chondroitinase ABC, and heparinase. The apical portions of the epithelial and glandular cells, obviously expanded by the estrogen treatment, display strong beta-metachromasia with TB (pH 4.0), saliva-resistant PAS-positive reactions, and also alcianophilia with AB (pH 2.5). These reactions are not reduced after the treatment with the enzymes above-mentioned. Meanwhile, in the stromal matrix, the same enzymes give an influence to diminish the reactions to various extent. Our results suggest that the estrogen-induced sulfated glycoprotein is definitely localized in the apical portions of the epithelial and glandular cells. The identity is emphasized between the substance that is elucidated in the histochemical sections and the sulfated glycoproteins that have been specified solely by means of biochemical assays.     

Effect of rotavirus strain on the murine model of biliary atresia

Biliary atresia is a devastating disorder of the newborn in which afflicted infants develop inflammation and fibrosis of the extrahepatic biliary tract, resulting in cirrhosis and end-stage liver disease. Infection with a virus is thought to be a contributing factor in the etiology of biliary atresia. In the murine model of biliary atresia, perinatal exposure to rhesus rotavirus (RRV) results in biliary epithelial cell infection causing bile duct obstruction. The purpose of this study was to determine if tropism for the biliary epithelial cell was unique to RRV. Newborn mice underwent intraperitoneal injection with five strains of rotavirus: RRV (simian), SA11-FM (simian/bovine), SA11-SM (simian), EDIM (murine), and Wa (human). RRV and SA11-FM caused clinical manifestations of bile duct obstruction and high mortality. SA11-SM caused clinical signs of hepatobiliary injury but the mortality was markedly reduced. EDIM and Wa caused no sign of hepatobiliary disease. The systemic and temporal distribution of viral protein and live virus varied according to the injected strain. Immunohistochemistry revealed that RRV and SA11-FM targeted the biliary epithelial cells. In contrast, SA11-SM was found in the liver but in not in the biliary epithelium. These results indicate that strain-specific characteristics dictate tropism for cells of hepatobiliary origin which in turn impact the ability to induce the murine model of biliary atresia.     

Extinction of passive avoidance response in various mouse strains

Dependence of the passive avoidance extinction dynamics on a mouse strain was shown. Mice C57BL/6J and AKR/J extinguished more quickly relative to DBA/2J, CBA/Lac and BALB/c, and this extinction was stable. Individual instability of extinction was characteristic of C3H/HeJ mice. Extinction of the passive avoidance in mice CBA/Lac and BALB/c was slower: with a delay in the beginning and prolonged retention of memory trace of the shock exposure. In DBA/2J mice, the extinction was impaired. These data suggest that DBA/2J, CBA/Lac and BALB/c mice constitute groups of risk with high predisposition to impairment of extinction of memory of aversive events, which is thought to be a symptom of a depressive-like state.