Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows.

Our objectives were to evaluate the effects of complete replacement of inorganic salts of trace minerals (STM) with organic trace minerals (OTM) in both pre- and postpartum diets on ovarian dynamics, estrous behavior measured by sensors, preimplantation conceptus development, and reproductive performance in dairy cows. Pregnant cows and heifers (n = 273) were blocked by parity and body condition score and randomly assigned to either STM or OTM diets at 45 ± 3 d before their expected calving. Pre- and postpartum diets were formulated to meet 100% of recommended levels of each trace mineral in both treatments, taking into consideration both basal and supplemental levels. The final target concentrations of Co, Cu, Mn, Se, and Zn were, respectively, 0.25, 13.7, 40.0, 0.3, and 40.0 mg/kg in the prepartum diet, and 0.25, 15.7, 40.0, 0.3, and 63.0 mg/kg in the postpartum diet. The STM group was supplemented with Co, Cu, Mn, and Zn sulfates and sodium selenite, while the OTM group was supplemented with Co, Cu, Mn, and Zn proteinates and selenized yeast. Treatments continued until 156 d in milk (DIM) and were assigned to individual cows using automatic feeding gates. Starting at 21 DIM, ultrasonography examinations of the ovaries were performed weekly to determine the presence of a corpus luteum and postpartum resumption of ovarian cyclicity. Cows were presynchronized with 2 injections of PGF2α at 42 and 56 DIM. Estrous behavior was monitored using electronic activity tags that indirectly measured walking activity. Cows detected in estrus after the second PGF2α were inseminated, and those not detected in estrus by 67 DIM were enrolled in a synchronization program. Cows that returned to estrus after artificial insemination (AI) were reinseminated. Pregnancy diagnosis was performed 33 d after AI, and nonpregnant cows were resynchronized. Transcript expression of interferon-stimulated genes in peripheral blood leukocytes was performed in a subgroup of cows (STM, n = 67; OTM, n = 73) on d 19 after AI. A different subgroup of cows (28 STM, 29 OTM) received uterine flushing 15 d after AI for recovery of conceptuses and uterine fluid for analyses of transcriptomics and metabolomics, respectively. In addition, dominant follicle diameter, luteal size and blood flow, and concentration of progesterone in plasma were measured on d 0, 7, and 15 relative to AI. After flushing, PGF2α was given and the dominant follicle was aspirated 2 d later to measure the concentration of trace minerals by mass spectrometry. Estrous behavior, size of the dominant follicle and corpus luteum, concentration of progesterone, time to pregnancy, and proportion of cows pregnant by 100 d of the breeding period did not differ between treatments. A greater proportion of cows supplemented with OTM had a corpus luteum detected before presynchronization (64.3 vs. 75.2%), and primiparous cows supplemented with OTM tended to resume cyclicity earlier than their STM counterparts. Cows supplemented with OTM had a greater concentration of Cu in follicular fluid than cows supplemented with STM (0.89 vs. 0.77 µg/mL, respectively). In pregnant multiparous cows, expression of receptor transporter protein 4 in peripheral blood leukocytes was 42% greater in the OTM group. Conceptuses of the 2 treatments had 589 differentially expressed transcripts, with many indicating advanced conceptus elongation and greater transcript expression of selenoproteins in the OTM group. In pregnant cows, 24 metabolites were more abundant in the uterine fluid of OTM, including spermidine, sucrose, and cholesterol. In conclusion, replacing STM with OTM caused modest improvements to resumption of ovarian cyclicity and important changes in preimplantation conceptus development, but it did not alter conception risk and pregnancy rate.

PIWIs, piRNAs and Retrotransposons: Complex battles during reprogramming in gametes and early embryos.

Gamete and embryo development are indispensable processes for successful reproduction. Cells involved in these processes acquire pluripotency, the ability to differentiate into multiple different cell types, through a series of events known as reprogramming that lead to profound changes in histone and DNA methylation. While essential for pluripotency, this epigenetic remodelling removes constraints that normally limit the expression of genomic sequences known as transposable elements (TEs). Unconstrained TE expression can lead to many deleterious consequences including infertility, so organisms have evolved complex and potent mechanistic arsenals to target and suppress TE expression during reprogramming. This review will focus on the control of transposable elements in gametes and embryos, and one important TE suppressing system known as the PIWI pathway. This broadly conserved, small RNA-targeted silencing mechanism appears critical for fertility in many species and may participate in multiple aspects of gene regulation in reproduction and other contexts.

Telomere length and telomerase activity in bovine pre-implantation embryos in vitro.

Telomeres are specialized structures that cap the ends of chromosomes and help to maintain genomic integrity and stability. Telomeres undergo dynamic changes during embryo development, which also represents an important stage for telomere elongation through telomerase enzyme activity. The objectives of this study were to examine changes in telomere length and telomerase activity from the early oocyte, through to the blastocysts stage of development, and the expression of factors with the potential to directly regulate telomeres. In vitro-produced bovine embryos were lysed and analysed for either relative telomere length, or telomerase activity using quantitative real-time PCR protocols. Our results reveal that relative telomere length is the shortest in the presumptive zygote stage of development and gradually increases to the blastocyst stage. We also demonstrate that differences between the mean telomere lengths throughout these stages are statistically significant (p < 0.05). Telomerase activity in the stages examined appears relatively constant until the blastocyst, where the highest level of activity is detected, leading to a significant difference in telomerase activity across embryonic stages (p < 0.005). Bovine telomerase RNA component (bTERC) expression levels were highest in the blastocyst, TERF1 transcripts showed little change in expression, and TERF2 expression decreased in the blastocysts (p < 0.05). Our results suggest that a complex integration of telomere-related RNA and proteins influences the regulatory mechanisms involved in 'reprogramming' of telomeres during early embryonic stages.

Large-scale vibrating coil magnetometer for the magnetic characterization of bulk superconductors.

This work presents the design and validation of a vibrating coil magnetometer for the characterization of the field dependence of the critical current density of centimeter-sized bulk superconductors as an alternative to the destructive methods typically used. The magnetometer is also shown to be capable of measuring the magnetic moment in an applied field of up to 5 T for diverse magnetic materials, such as soft and hard ferromagnets and high-temperature superconducting pellets. The vibrating coil magnetometer was first optimized using finite element simulations and calibrated using a commercial vibrating sample magnetometer. The vibrating coil magnetometer was benchmarked with hysteresis measurements of a Nd2Fe14B disk made with a commercial hysteresisgraph, showing good agreement between the different setups. The magnetic hysteresis of a YBa2Cu3O7-x superconducting pellet was measured at 77 K, showing a penetration field of 1 T and an irreversibility field of 4 T. The field dependent critical current density of the superconductor was then inferred from the magnetic hysteresis measurements and extrapolated at low fields. Finally, the resulting critical current density was used to successfully reproduce the measured magnetization curve of the pellet at 2 T with finite element simulations.

Regulation of macrophage alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein by lipopolysaccharide and interferon-gamma.

alpha 2-Macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2M-R/LRP) is a broad specificity receptor that may function in lipoprotein metabolism, proteinase regulation, and growth factor regulation. In this study, we demonstrated that alpha 2M-R/LRP expression in macrophages can be markedly decreased by LPS and by IFN-gamma. Regulation of alpha 2M-R/LRP in RAW 264.7 cells was demonstrated at the mRNA, antigen, and receptor-function levels. In receptor-function studies, the decrease in alpha 2M-R/LRP expression was detected as a 90% decrease in the Bmax or maximum receptor binding capacity for activated alpha 2M after treatment with LPS or IFN-gamma. Western blot analysis of whole cell lysates demonstrated significant loss of alpha 2M-R/LRP heavy-chain. Northern blot analysis of poly(A)+ RNA revealed a marked decrease in alpha 2M-R/LRP mRNA after treatment with LPS (79% decrease) or IFN-gamma (70% decrease). Other cytokines, including tumor necrosis factor-alpha, transforming growth factor-beta-1, and interleukin-6 did not regulate alpha 2M-R/LRP. The ability of LPS and IFN-gamma to regulate alpha 2M-R/LRP was confirmed in experiments with primary cultures of murine bone marrow macrophages. These studies demonstrate that macrophage alpha 2M-R/LRP is subject to significant downregulation by physiologically significant cytokines and signaling macromolecules.

An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice.

Radioiodinated transforming growth factor-beta 1 (TGF-beta 1) bound to the plasma proteinase inhibitor, alpha 2-macroglobulin (alpha 2M), as determined by chromatography on Superose-6 and native polyacrylamide gel electrophoresis. When alpha 2M conformational change was induced with methylamine, 125I-TGF-beta 1 binding significantly increased. Intravenously injected 125I-TGF-beta 1 cleared from the circulation of mice rapidly at first; however, intravascular radioactivity stabilized near 20% of the initial level. At necropsy, radioactivity was recovered predominantly in the liver (65%); however, the density of radioactivity (disintegrations per minute/g organ wt) was highest in the lungs. Markedly different results were obtained with purified 125I-TGF-beta 1-alpha 2M-methylamine complex. Clearance of the complex occurred as a first-order process with a t1/2 of 4 min. Greater than 90% of the radioactivity was recovered in the liver. The clearance and distribution of 125I-TGF-beta 1-alpha 2M-methylamine were equivalent to those observed with 125I-alpha 2M-methylamine and 125I-alpha 2M-trypsin. The latter two radioligands clear via specific alpha 2M receptors in the liver. Large molar excesses of alpha 2M-trypsin or alpha 2M-methylamine competed with 125I-TGF-beta 1-alpha 2M-methylamine for plasma clearance. Native alpha 2M, which does not bind to the alpha 2M receptor, did not compete. The receptor binding domain of alpha 2M-methylamine was blocked by chemical modification or enzyme treatment. The resulting alpha 2M preparations still bound 125I-TGF-beta 1; however, the complexes did not clear when injected intravenously in mice. The studies presented here demonstrate that alpha 2M can mediate the plasma clearance of a growth factor via the alpha 2M receptor system. We propose that alpha 2M, the alpha 2M receptor, and proteinases may function as a concerted system to regulate TGF-beta 1 activity and the activity of related factors in vivo.

Expression of microRNAs miR-21 and miR-181c in cerebrospinal fluid and serum in canine meningoencephalomyelitis of unknown origin.

The potential of microRNAs (miRNAs) as biomarkers for canine meningoencephalomyelitis of unknown origin (MUO) was investigated by using quantitative real-time (qRT)-PCR to determine the expression of microRNA-21 (miR-21) and microRNA-181c (miR-181c) in the cerebrospinal fluid (CSF) of dogs. Dogs with MUO (n = 10) had higher levels of expression of miR-21 and miR-181c in the CSF than dogs with non-inflammatory neurological diseases (n = 8). There was a positive correlation between CSF cellularity and expression of miRNAs in the CSF, particularly for miR-21 in the MUO group.

Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2.

The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-beta and native or reacted forms of alpha 2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native alpha 2M with plasmin or methylamine markedly increased the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to alpha 2M. The alpha 2M-plasmin/TGF-beta complexes were minimally dissociated by heparin. Reaction of alpha 2M with thrombin or trypsin reduced the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-beta 2 and native or reacted forms of alpha 2M were less dissociable by heparin than the equivalent complexes with TGF-beta 1. These studies demonstrate that the TGF-beta-binding activity of alpha 2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that alpha 2M-plasmin preferentially binds TGFs-beta suggest a mechanism by which alpha 2M may regulate availability of TGFs-beta to target cells in vivo.

Transcriptional regulation of LDL receptor-related protein by IFN-gamma and the antagonistic activity of TGF-beta(1) in the RAW 264.7 macrophage-like cell line.

Low density lipoprotein receptor-related protein (LRP) is a major receptor for multiple ligands, including chylomicron and VLDL remnants, bacterial toxins, viruses, proteinases, lipoprotein lipase, and activated alpha2-macroglobulin (alpha2M). In this study, we used Northern blot analyses and nuclear run-on experiments to demonstrate that interferon-gamma (IFN-gamma) causes a concentration-dependent decrease in steady-state LRP mRNA expression and gene transcription rate in RAW 264.7 cells. IFN-gamma also markedly increased expression of inducible nitric oxide synthase (NOS), as expected; however, the increase in nitric oxide was not responsible for the down-regulation of LRP expression since the NOS inhibitor, N(G)-monomethyl-L-arginine, did not preserve LRP expression in IFN-gamma-treated cells. Transforming growth factor-beta1 (TGF-beta1; 2.5 ng/mL) had no independent effect on LRP expression and did not modify the response to IFN-gamma when the two cytokines were added simultaneously to cultures. When TGF-beta1 was added 24 h prior to IFN-gamma, the extent of LRP down-regulation was significantly reduced. Specific binding of the LRP ligand, activated (125)I-alpha2M, was decreased by 76 +/- 5% in cells treated with 100 U/mL IFN-gamma, but only by 45 +/- 7% in cells treated with 100 U/mL IFN-gamma after TGF-beta1-pretreatment. The antagonistic activity of TGF-beta1 on the IFN-gamma response in RAW 264.7 cells did not result from a change in LRP mRNA stability or IFN-gamma receptor expression, as determined by Northern blot analyses and (125)I-IFN-gamma binding experiments. The studies presented here suggest that the balance between IFN-gamma and TGF-beta1 may be critical in determining LRP expression at sites of infection and inflammation.

Alpha 2-macroglobulin conformation determines binding affinity for activin A and plasma clearance of activin A/alpha 2-macroglobulin complex.

Activin A is a member of the transforming growth factor-beta family of growth factors and a potent regulator of cellular activity. A number of binding proteins for activin A have been identified, including alpha 2-macroglobulin (alpha 2M). Alpha 2M has several conformational states that are known to have different growth factor-binding properties. The effect of alpha 2M conformation on activin A binding has not been characterized. The aims of this study were to determine 1) whether activin A binds preferentially to the native (alpha 2M-N) or "activated" (alpha 2M*) conformation of alpha 2M, 2) the affinity of different alpha 2M conformations for activin A, and 3) the fate of activin A complexed with alpha 2M-N or alpha 2M* in vivo. [125I]Activin A associated with alpha 2M in plasma and follicular fluid and with purified alpha 2Ms. In this qualitative assay, more activin A was associated with alpha 2M* than with alpha 2M-N. The affinity of the activin A-alpha 2M interaction was determined. The Kd values for activin A-alpha 2M* and activin A-alpha 2M-N were 190 +/- 30 and 510 +/- 60 nM, respectively. The plasma clearance profiles and tissue distribution of uncomplexed activin A and purified alpha 2M*-activin A complex were determined. Radiolabeled activin A cleared in a biphasic manner, with rapid clearance over the initial 10 min and substantially slower clearance over the subsequent 20 min. During the slow phase of clearance, activin A formed a complex with circulating alpha 2M-N. In contrast, radiolabeled activin A-alpha 2M* complexes were rapidly cleared from plasma with a half-life of approximately 5 min and were specifically targeted to alpha 2M receptors in vivo. These studies reveal that alpha 2M can maintain activin A in the circulation or rapidly target the hormone for plasma clearance depending on the conformational state of the carrier protein in vivo.

Pseudo type III dyslipoproteinemia is associated with normal fibroblast lipoprotein receptor activity.

Pseudo type III (PT-III) dyslipoproteinemia is characterized by a plasma accumulation of triglyceride-rich lipoproteins (TRL) and their remnants. It mimics type III, but its etiology can not be ascribed to a genetic apo E defect. In order to determine whether PT-III is associated with a genetic lipoprotein receptor abnormality, we have measured (in cultured fibroblasts from affected and nonaffected individuals) the in vitro activity of three lipoprotein receptors which are implicated in the catabolism of TRL, namely the low-density lipoprotein receptor (LDL-R), the lipoprotein receptor-related protein (LRP) and the lipolysis-stimulated receptor (LSR). Specific cell association and degradation of 125I-LDL by LDL-R-upregulated PT-III fibroblasts was not significantly different from that of control cells (103 +/- 10% and 98 +/- 17% of controls; 20 microg/ml 125I-LDL). Specific cell association and degradation of rabbit 125I-beta-VLDL was also not significantly different. LRP activity was assessed by measuring the ability of PT-III and control cells to bind three different LRP ligands: activated alpha2-macroglobulin (alpha2M-MA), lactoferrin and apo E-enriched rabbit beta-VLDL. No significant differences were observed (24.0 +/- 2.1 vs. 23.4 +/- 5.7 fmol/mg for 5 nM of 125I-alpha2M-MA; 4.8 +/- 0.3 vs. 5.2 +/- 1.3 microg/mg for 20 microg/ml of 125I-lactoferrin; 319.4 +/- 51.2 vs. 309.5 +/- 23.2 ng/mg for 5 microg/ml of 125I-beta-VLDL, PT-III vs. control, respectively). LSR activity, as assessed by the cell association or degradation of 125I-LDL by fibroblasts in the presence of 0.5 mM oleate and human leptin, was also not different. No evidence was obtained for deficient cellular recognition of PT-III TRL (d < 1.006 g/ml) by normal human fibroblasts or mouse macrophages. These results suggest that PT-III dyslipoproteinemia is not due to an accumulation in plasma of poorly recognized TRL, nor due to a genetic defect in LDL-R, LRP or LSR.

Identification of a 43-kDa protein in human liver cytosol that binds to the 3'-untranslated region of CYP2A6 mRNA.

Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 has not been established. The murine orthologue Cyp2a5 is regulated post-transcriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3'-untranslated region (3'-UTR) of Cyp2a5 mRNA has been identified, but its role in mRNA stabilization is unclear. We hypothesized that similar interactions occur between cytosolic proteins in human liver and CYP2A6 3'-UTR mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepatic cytosolic protein that binds specifically to sequences in the 3'-UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminated with proteinase K digestion. Complex formation was inhibited with a molar excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Protein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linking of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes corresponding to various lengths of CYP2A6 mRNA, the protein-binding site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3'-UTR. Complex formation with hepatic cytosolic protein from four human subjects correlated with levels of hepatic CYP2A6 microsomal protein, suggesting a possible regulatory role. Further characterization of the RNA-binding protein, the primary binding site, and the influence of this interaction on CYP2A6 mRNA stability will help to elucidate the relevance of these findings to the post-transcriptional control of CYP2A6.

Human ovarian follicular fluid has functional systems for the generation and modulation of thrombin.

OBJECTIVE: To determine whether prothrombin is present in follicular fluid and whether the enzymatic pathways for prothrombin activation are similar to those in plasma. DESIGN: Follicular fluid samples collected at the time of oocyte harvest for an assisted reproductive technology procedure (ART) were analyzed for a panel of hemostatic proteins with use of a combination of functional, chromogenic, and Western ligand blot analysis. SETTING: An ART clinic and an academic research laboratory. PATIENT(S): Women undergoing ART. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Determination of components of thrombin generation and thrombin modulatory systems using functional and antigenic assay procedures. RESULT(S): Both prothrombin and components of the prothrombinase enzyme complex, which includes factors V, VII, and X, are present in follicular fluid. Other hemostatic proteins, including factors VIII and IX and vonWillebrand factor, are absent. The direct activation of prothrombin to thrombin is similar in follicular fluid and plasma. Like plasma, inhibitors of both thrombin and thrombin generation, including antithrombin, protein C, and alpha2-macroglobulin, are present in follicular fluid. CONCLUSION(S): Only a select group of hemostatic plasma proteins are present in follicular fluid. There is no direct correlation between molecular size and concentration of individual proteins in follicular fluid. These results indicate that the proteins involved in the thrombin-generating and thrombin modulatory pathways may be derived from ovarian cells, suggesting that thrombin may have a role in folliculogenesis.

Divergent Pseudomonas exotoxin A sensitivity in normal and transformed liver cells is correlated with low-density lipoprotein receptor-related protein expression.

Pseudomonas exotoxin A (PEA) is an extracellular virulence factor produced by the opportunistic human pathogen Pseudomonas aerguinosa. PEA intoxification begins when PEA binds to the low-density lipoprotein receptor-related protein (LRP). The liver is the primary target of systemic PEA, due largely to the high levels of functional LRP expressed by liver cells. Using a 3H-leucine incorporation assay to measure inhibition of protein synthesis we have demonstrated that normal (BNL CL.2) and transformed (BNL 1ME A7R.1) liver cells exhibit divergent PEA sensitivity; with BNL 1ME A7R.1 cells demonstrating greater PEA sensitivity than their non-transformed counterparts. The receptor-associated protein, a LRP antagonist, decreased PEA toxicity in BNL 1ME A7R.1 cells, confirming the importance of the LRP in PEA intoxification in this cell type. Increased PEA sensitivity in BNL 1ME A7R.1 cells was associated with increased functional cell surface LRP expression, as measured by alpha2-macroglobulin binding and internalization studies, and increased LRP mRNA levels, as determined by Northern blot analysis. Interestingly, BNL CL.2 cells were more sensitive than BNL 1ME A7R.1 cells to conjugate and mutant PEA toxins that do not utilize the LRP for cellular entry. These data demonstrate that increased LRP expression is an important mechanism by which PEA sensitivity is increased in BNL 1ME A7R.1 transformed liver cells.

Amounts of selected coagulation factors in pre- and post-mortem follicular fluid are similar and do not correlate with molecular mass.

This study was designed to evaluate the amounts of coagulation factors and to determine whether the protein profile in pre-ovulatory ovarian follicular fluid aspirated from ovaries collected from mares at slaughter are representative of that in follicular fluid collected from live animals. The proteins evaluated included, (i) albumin, ceruloplasmin and fibronectin, (ii) the procoagulant plasma proteins, Factor V (FV), Factor VII (FVII), Factor X (FX) and prothrombin, and (iii) the anticoagulant plasma proteins, antithrombin and alpha2-macroglobulin. The amounts of the individual proteins were similar in both types of follicular fluid. There was no correlation between the activity of FV, FVII, FX or prothrombin in follicular fluid and their molecular size although a correlation was found for the other proteins. These results suggest that the procoagulant proteins in follicular fluid are not likely derived from plasma. The total protein content of follicular fluid samples collected from both sources was similar and the results determined with the Biuret, Lowry and Biorad methods were also not significantly different (P>0.05).

The functional independence of mands and tacts.

This study demonstrates functional independence in the acquisition of mands and tacts. Some subjects first learned to mand the experimenter's placement of objects with the prepositional phrases "On the left" and "On the right." They were regularly tested for collateral appearance of tacts with these same phrases. Other subjects learned to tact the location of objects with these prepositional phrases and were regularly tested for collateral appearance of mands. All subjects were next trained in the repertoire that had not been trained in the first condition (either tact or mand). After all subjects had learned both to mand and to tact correctly, another assessment of mand-tact independence was undertaken. Mands (tacts) were reversed and testing assessed collateral reversal of tacts (mands). The results demonstrated that tacts and mands, even when incorporating identical response forms, were functionally independent during acquisition. Subsequent modification of one repertoire (by reversal training) produced collateral reversal in three of nine subjects.

Macrophage populations and apoptotic cells in the liver before spontaneous hepatitis in Long-Evans Cinnamon (LEC) rats.

The inbred mutant strains of Long-Evans Cinnamon (LEC) rats spontaneously develops acute hepatitis as a result of abnormal copper accumulation, followed by chronic hepatitis, cholangiofibrosis and hepatocellular carcinoma. To shed some light on the role of macrophages in the liver failure, immunohistochemical methods were used to investigate the kinetics of macrophage populations in the liver of male LEC rats, in relation to the appearance of myofibroblastic cells and hepatocyte apoptosis. Rats examined at 24 weeks of age and moribund rats killed at 22-25 weeks of age had increased serum concentrations of aspartate aminotransferase and alanine aminotransferase, with jaundice and histological changes indicative of hepatic failure, whereas rats examined at 8, 12, 16 or 20 weeks old showed no such abnormal findings. Immunolabelling with ED1 (a monoclonal antibody recognizing rat macrophages) and ED2 (a monoclonal antibody specific for rat resident macrophages) revealed that numbers of blood monocyte-derived macrophages and Kupffer cells began to increase markedly at 16 weeks of age (before the onset of hepatitis). However, alpha-smooth muscle actin (SMA)-positive myofibroblastic cells (modulated perisinusoidal cells) and hepatocyte apoptosis, demonstrable by the TUNEL method, were rarely seen at 8, 12, 16, 20 or 24 weeks. There was no close relationship between macrophage expansion and the appearance of myofibroblastic cells or hepatocyte apoptosis. In moribund rats, only a few SMA-positive cells were seen in the periportal zones; hepatocytes undergoing apoptosis increased in number, and macrophages engulfing apoptotic bodies were observed occasionally, suggesting that apoptosis was related to hepatic failure as an early event. In addition, immunohistochemical examination demonstrated abnormal deposits of laminin along the sinusoids from 20 weeks, as an initial extracellular matrix protein in LEC rat livers.

Effects of lipopolysaccharide on a macrophage-like cell line (HS-P) from a rat histiocytic sarcoma.

Lipopolysaccharide (LPS) is a major modulator of macrophage functions. To characterize a newly established rat histiocytic sarcoma-derived cell line (HS-P), immunophenotypic changes and cellular growth responses of HS-P cells exposed to LPS were investigated and compared with those of MT-9 cells isolated from a rat malignant fibrous histiocytoma. MT-9 cells have somewhat histiocytic features, because occasional cells react to rat macrophage-specific antibodies. Addition of LPS to cultured HS-P cells increased the numbers of cells immunopositive to ED1 (rat macrophage-specific antibody) and ED2 (rat histiocyte-specific antibody) and stimulated the phagocytosis of latex beads, whereas LPS-treated MT-9 cells did not show such immunophenotypic changes. LPS-treated HS-P cells showed enhanced immunolabelling of alpha-smooth muscle actin, suggesting a possible modulation of macrophages towards myofibroblastic cells. To evaluate cellular growth after the addition of LPS or fetal bovine serum, DNA synthesis was examined by measuring tritiated thymidine incorporation, and the mRNA expression of c- jun and c- myc (immediate early genes in the cell cycle) was examined by Northern blot analysis. In HS-P cells, the addition of serum greatly increased DNA synthesis and induced high expression of c- jun and c- myc; in contrast, LPS markedly depressed DNA synthesis and reduced the expression of c- jun and c- myc. HS-P cells were more sensitive than MT-9 cells to the growth-promoting effect of serum and the growth-inhibiting effect of LPS. The study demonstrated that HS-P cells are highly LPS-responsive, indicating that they would be useful for studies of macrophage functions.

Influence of progesterone and oestrogen on growth and morphology of a transplantable rat uterine smooth muscle tumour (SMT-Y).

Tumours of uterine smooth muscle are poorly understood neoplasms in which the effects of steroid sex hormones are complex. The influence of progesterone and oestrogen on a transplantable rat uterine smooth muscle tumour line (SMT-Y) was investigated. Female F344 rats given subcutaneous transplants of tumour fragments developed tumours, 1.5-2 cm in diameter, and were then treated with progesterone (10 mg/rat) or 17 beta-oestradiol (50 mg/rat). Tumours in treated groups were compared with those in untreated controls. During a 9-week observation period after treatment, progesterone promoted tumour growth from 4 weeks, with increased numbers of proliferating cells. In contrast, oestradiol inhibited tumour growth from 6 weeks; the degraded tumours, consisting mainly of vacuolated neoplastic cells, had decreased numbers of proliferating cells and increased numbers of apoptotic cells, demonstrable by in-situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick labelling. Immunohistochemically, tumours in control and progesterone groups were labelled positively for alpha-smooth muscle actin (SMA) and desmin but not for vimentin, whereas the degraded tumours in the oestradiol group had reduced reactivity for SMA and desmin but an increased reactivity for vimentin. These results indicate that progesterone may act as a promoter for uterine smooth muscle tumour growth by stimulating mitotic activity, whereas oestrogen may have suppressive effects on tumour growth, accompanied by morphological changes.

Macrophage-like cell line (HS-P) from a rat histiocytic sarcoma.

With future exploration of macrophage properties in mind, we established a novel cell line (HS-P) from a transplantable histiocytic sarcoma, derived originally from a tumour in an aged F344 rat. HS-P was subjected to 70 serial passages, in which the mean doubling time was 15.7 h. The cells, which were round, oval or polygonal in shape, were arranged in a compact sheet. They reacted to varying degrees for lysosomal enzymes (acid phosphatase and non-specific esterase) and with the following antibodies: ED1/ED2 (rat macrophage/histiocyte-specific), OX6 (rat MHC class II-specific), lysozyme antibody and alpha1-antichymotrypsin antibody. Electron microscopically, HS-P cells showed lysosomes and prominent cell projections. These findings indicated that the cultured cells were macrophage-like. Syngeneic rats inoculated subcutaneously or intraperitoneally with HS-P cells invariably developed sarcomatous tumours consisting of monomorphic mononuclear cells, which exhibited cytochemical properties similar to those of cultured HS-P cells. Bioassay and reverse transcription-polymerase chain reaction methods revealed that tumour necrosis factor-alpha increased on addition of lipopolysaccharide (LPS), indicating that HS-P cells remained LPS-responsive. HS-P cells may prove to be a useful tool for in-vitro studies of macrophage function.