Effect of Losartan, an Angiotensin II Antagonist, on Hepatic Fibrosis Induced by CCl4 in Rats

In addition to regulating blood pressure and body fluid homeostasis, the renin-angiotensin system (RAS) is also involved in hepatic fibrogenesis. We aimed to investigate the effect of losartan, an angiotensin II (Ang II) antagonist, on CCl4-induced hepatic fibrosis in rats. Hepatic fibrosis was induced by a subcutaneous injection with 50% CCl4 in Sprague-Dawley rats. The amount of CCl4 administered was 1 mg/kg. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma and hydroxyproline (Hyp) contents in liver tissue were assayed by spectrophotometry. Hyaluronic acid (HA) and procollagen III (PC III) were assessed by radioimmunoassay. Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) levels in culture supernatants of Kupffer cells (KCs) stimulated with Ang II was determined by ELISA. Liver samples collected after 12 weeks of CCl4 treatment were stained with hematoxylin and eosin, then scored. Losartan (2.5, 5, and 10 mg x kg(-1), ig) and captopril (100 mg x kg(-1), ig) significantly decreased liver and spleen indexes, serum transaminase (AST, ALT) activities, HA and PC III levels, and Hyp contents in liver tissue in rats of hepatic fibrosis. Histopathological scores showed that losartan had an inhibitory effect on the progression of hepatic fibrosis. In in vitro experiments, losartan (1 x 10(-9) - 1 x 10(-5) M) significantly reduced TNF-alpha and TGF-beta1 levels in culture supernatants of KCs, but captopril (1 x 10(-5) M) did not. The results showed that losartan significantly inhibited the progression of hepatic fibrosis induced by CCl4, and the inhibitory effect of losartan on hepatic fibrosis might be associated with its ability to inhibit the production of TNF-alpha and TGF-beta1 by activated KCs.     

Effects of Chronic Administration with Nilvadipine Against Immunohistochemical Changes Related to Aging in the Mouse Hippocampus

We investigated the effect of Ca²⁺ antagonist nilvadipine on age-related immunohistochemical alterations in ubiquitin and S100 protein of the hippocampal CA1 sector in mice using 8-, 18-, 40-, and 59-week-old mice. No significant changes in the number of neuronal cells were observed in the hippocampal CA1 sector up to 59 weeks after birth. The administration of nilvadipine did not affect the number of the hippocampal CA1 cells of 40-week-old mice. Age-dependent increases in ubiquitin immunoreactivity were observed in the hippocampal CA1 neurons up to 59 weeks after birth. The administration of nilvadipine prevented dose-dependently the increases in the number of ubiquitin-immunoreactive neurons in the hippocampal CA1 sector of 40-week-old mice. S100 immunoreactivity was unchanged in the hippocampal CA1 sector up to 40 weeks after birth. In 59-week-old mice, the level of staining of S100-immunoreactive cells increased significantly in the hippocampal CA1 sector. The administration of nilvadipine decreased dose-dependently the number of S100-immunoreactive cells in the hippocampal CA1 sector of 40-week-old mice. The present study demonstrates that age-related increases in ubiquitin system may play a pivotal role in protecting neuronal cell damage during aging. In contrast, our results suggest that expression of S100 protein in the hippocampal CA1 sector may play an exacerbating factor in some neuronal cells damaged by aging. Our results also demonstrate that nilvadipine, a dihydropyridine-type calcium channel blocker, can prevent dose-dependently the increases in the ubiquitin immunoreactive neurons and decrease the number of S100 immunoreactive cells in the hippocampal CA1 neurons of aged mice. These results suggest that nilvadipine may offer a new approach for the treatment of neuronal dysfunction in aged humans.     

Serum collagen type VI and XIV and hyaluronic acid as early indicators for altered connective tissue turnover in alcoholic liver disease

Hepatic fibrosis in alcoholic liver disease often heralds progression to cirrhosis and, therefore, noninvasive parameters are required for early diagnosis and follow-up. Collagens VI and XIV, procollagen-III-N-propeptide, hyaluronic acid, and active transforming growth factor-₁ (TGF-₁) were measured in healthy volunteers, patients with alcoholic cirrhosis, and heavy drinkers without cirrhosis. Noncirrhotic alcoholics were assigned to two groups with either normal aspartate aminotransferase or levels 2 normal. Collagens VI and XIV were elevated in all alcoholic patients compared to controls (P < 0.0001, all instances). Procollagen-III-N-propeptide and hyaluronic acid levels were higher in alcoholic patients with elevated liver enzymes and in cirrhotics as compared to controls. Procollagen-III-N-propeptide revealed a significant correlation with serum levels of TGF-₁ (P < 0.0001). Collagens VI, and XIV, procollagen-III-N-propeptide, and hyaluronic acid appear to be sensitive markers indicating fibrotic transformation in alcoholics. The correlation between procollagen-III-N-propeptide and TGF-₁ emphasizes its role in hepatic fibrogenesis.     

GDF-9 and BMP-15: Oocyte Organizers

It has been long known that oocyte-secreted factors play key roles in development and differentiation of ovarian follicles, but the factors involved in folliculogenesis are not well characterized to date. The recent studies on two novel TGF superfamily members, GDF-9 and BMP-15, have dramatically changed our view of ovarian physiology. GDF-9 has been shown to be obligatory for fertility in female mice by loss-of-function studies; in vitro studies further determined that GDF-9 is essential at multiple steps in the process of female reproduction. BMP-15, which shares high homology with GDF-9, seems not as essential as GDF-9 in fertility of female mice, though its expression pattern resembles GDF-9 quite closely. However, studies on sheep carrying natural mutations in the Bmp15 gene, FecX^I and FecX^H, show that BMP-15 is associated with infertility and super-fertility in a dosage-sensitive manner. Evidence from recent in vitro experiments also indicates that BMP-15 inhibits major FSH actions that are obligatory for follicle development and ovulation by suppressing FSH receptor expression in rats. Taken together, GDF-9 and/or BMP-15 may play dominant roles in female fertility in a species-dependent manner. Further studies to clarify the mechanisms by which GDF-9 and BMP-15 function will help us understand whether GDF-9, BMP-15, or both are essential for human fertility.     

Effects of Pregnancy in Rats on Cysteamine-Induced Peptic Ulcers (Role of Progesterone)

After mating with a sexually active male, groupsof female Sprague-Dawley rats were injected withcysteamine (400 mg/kg, subcutaneously) at day 0(controls), day 5 (early-stage pregnancy), and day 18(late-stage pregnancy) of pregnancy. In contrast tolate-stage pregnancy rats, early-stage pregnancy animalsshowed a decrease of cysteamine-induced gastroduodenallesions. When subjected to cysteamine injection, both nonpregnant female and male rats treated foreight days with progesterone (300 g/rat,subcutaneously) showed a reduced incidence ofgastroduodenal lesions. No effect was found in animalspretreated with 17-estradiol (200 g/rat,subcutaneously). Furthermore, increased gastroduodenalmucus levels were found in early-stage pregnancy ratsand in animals pretreated with progesterone. Theseresults suggest that increased progesterone plasma levelsduring early-stage pregnancymay be involved inpregnancy-induced gastric and duodenal protection. Thiseffect may be related to an increase in gastric andduodenal mucus production induced by thishormone.     

Hemoglobin E and codon 17 nonsense: Two β-globin gene mutations common in Southeast Asia detected by the use of ARMS

Summary Hemoglobin E (codon 26 GAGAAG) and codon 17 nonsense (AAGTAG), two clinically important mutations of the -globin gene, are common in Southeast Asia. The detection of these mutations using allele-specific PCR is described. Together with the previously reported method for the detection of the common Southeast asian codon 41–42 frameshift mutation (del CTTT), it is possible to identify the vast majority of clinically important -globin gene mutations in Southeast Asian populations by means of nonradioactive methods.This study was supported by theVolkswagen-Stiftung     

Loss of Inhibitory Growth Regulation by TGF-β1 in Preneoplastic Lesions in Rat Liver

Injection of pig serum into rats twice a week for eight weeks induced transforming growth factor-₁ (TGF-₁) mRNA expression and protein production resulting in liver fibrosis without parenchymal cell injury. Eight-week treatment with pig serum reduced bromodeoxyuridine (BrdU) -positive hepatocytes 24 hr after 70% partial hepatectomy compared to that in the livers of rats treated with saline for eight weeks. Administration of a choline-deficient l-amino acid-defined (CDAA) diet for six weeks with pig serum coadministration, after pretreatment with pig serum for eight weeks, led to the development of preneoplastic lesions that were positive for the placental form of glutathione S-transferase (GSTP). Eight-week pretreatment with pig serum induced more GSTP-positive lesions and TGF-₁ mRNA expression and protein concentration in the livers of rats subsequently fed a CDAA diet for six weeks than in rats fed the CDAA diet with saline treatment. These results indicate that TGF-₁ induced by pig serum treatment inhibited hepatocyte proliferation but failed to prevent the development of preneoplastic lesions in a CDAA diet model.     

TCR1+ large granular lymphocyte proliferation in rheumatoid arthritis

The T-lymphoproliferative syndrome is characterized by a proliferation of large granular lymphocytes (LGL). It is often associated with neutropenia, and in 30% of cases with rheumatoid arthritis (RA). Phenotypic analysis has demonstrated that in most cases of RA with T-proliferative disease, the LGL represent T cells with a clonal rearrangement of the / T cell receptor (TCR2). Here, three patients with / TCR1⁺ LGL proliferation suffering from long-standing arthritis and neutropenia are described. The first patient with RA showed an expansion of a heterogeneous CD2⁺ CD16⁺ CD56^- LGL population, of which 30% coexpressed TCR1 with V1 rearrangement. The second patient with ankylosing spondylitis and RA was suffering from proliferation of TCR1⁺ (V9^-, V1^-), CD2⁺ CD16^- CD56^- LGL with low coexpression of CD8. The third patient with RA was suffering from a proliferation of TCR1⁺ (V1⁺, V9^-) CD4^- CE8^- CD16^- CD56^- lymphocytes. On the basis of these unusual findings, the pathogenetic role of TCR1⁺ T cells in RA is discussed.     

Hepatitis C virus replication is associated with expression of transforming growth factor-α and insulin-like growth factor-II in cirrhotic livers

The molecular role of hepatitis C virus (HCV) in liver disease has yet to be clarified. In this study, we analyzed the relationship of HCV replication with mRNA expression of growth factors and mutation of tumor suppressor gene, ie, transforming growth factor-₁ (TGF-₁), which promotes cirrhotic changes; TGF-, insulin-like growth factor-II (IGF-II), which are both related to hepatocyte transformation; and tumor suppressor genep53, which is associated with HCC progression. A semiquantitative RNA polymerase chain reaction (RNA-PCR) was used to analyze genetic expression in 31 cirrhotic liver specimens from patients with HCV. In order to detect HCV replication, the minus-strand RNA of HCV, which serves as a template for the synthesis of genomic plus-strand RNA, was examined. The expression of the growth factors was semiquantified by RNA-PCR, and the mutation ofp53 was detected using PCR-single-strand conformation polymorphism. According to the semiquantitative analysis, HCV replication was not associated with the expression of TGF-₁ but was significantly so with the overexpression of TGF- (r=0.74) and IGF-II (r=0.65) in the HCV-positive cirrhotic livers. No mutation ofp53 was recognized in any of the samples. Our investigation thus suggested that the replication of HCV might mediate the coexpression of TGF- and IGF-II and act as a possible initiating factor for hepatocarcinogenesis.     

Role of Central Interleukin-1β in Gastrointestinal Motor Disturbances Induced by Lipopolysaccharide in Sheep

Cytokines are involved in the symptoms of theacute phase response induced by infectious diseases inhumans as well as in animals, and interleukin-1(IL-1 ) has a pivotal role in these changes. The role of central IL-1 in the gastrointestinalhypomotility and fever evoked by intravenousadministration of lipopolysaccharide (LPS) and themechanisms involved, were investigated in sheep as anexperimental model. LPS (0.1 g/kg, intravenously)induced gastrointestinal hypomotility and fever thatwere significantly reduced by priorintracerebroventricular administration of IL-1receptor antagonist protein (IL-1ra, 2 g/kg). The effects of LPS were mimickedby intracerebroventricular IL-1 (50 ng/kg),whereas IL-1 injected intravenously at the samedose only caused a slight and transient fever withoutmodifying the gastrointestinal motility. Priorintracerebroventricular administration of thecyclooxygenase inhibitor indomethacin (100 g/kg) butnot the corticotropin-releasing factor (CRF) receptorantagonist -helical CRF_9-41 (5 g/kg) blocked alleffects evoked by both LPS and IL-1. These resultssuggest that in sheep, LPS induces digestive motordisturbances through a central release of IL-1 andprostaglandins.     

Chondrocyte phenotyping in human osteoarthritis

Cell-ECM (extracellular matrix) interactions are believed to play a key role in maintaining the normal structure of tissues such as cartilage. Cell surface adhesion molecules have been reported to mediate chondrocyte binding to ECM proteins in human normal cartilage but the behaviour of these molecules in human osteoarthritic cartilage is unknown. We studied receptor matrix proteins on freshly isolated chondrocytes obtained from 10 patients with osteoarthritis (OA). Chondrocytes were isolated by enzymatic digestion from three zones of the articular cartilage with a different degree of macroscopic and microscopic damage and chondrocyte phenotype was defined by flow cytometry. Chondrocytes strongly expressed ₁ integrin but not ₃ integrin. LFA-1 (CD18/CD11a) and ICAM-1 (CD54) antigens were almost undetectable. Interestingly, ₁ expression was significantly higher in the minimally damaged zone than in the zones with medium and maximum damage. These data show that ₁-integrin-mediated chondrocyte-ECM interactions decrease in osteoarthritic cartilage suggesting that perturbations of chondrocyte-matrix signalling occurs during OA.     

Overproduction of inhibitory hematopoietic cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells in patients with aplastic anemia

The aim of this study was to measure the level of cytokines produced by peripheral blood mononuclear cells (PBMNC) in patients with aplastic anemia (AA) and to determine their effect on the clonal growth of normal bone marrow (BM) cells. Twenty-one patients with AA and 11 normal controls were enrolled in this study. Medium conditioned by PBMNC of AA patients in the presence of lipopolysaccharide (LPS) was found to be suppressive to the colony growth of normal BM cells. Thus, we further determined the presence in the PBMNC-conditioned medium (CM) of both inhibitory cytokines: macrophage inflammatory protein-1 (MIP-1), tumor necrosis factor- (TNF-), transforming growth factor-2 (TGF-2), and interferon- (IFN-), and stimulatory cytokines: interleukin-3 (IL-3) and stem cell factor (SCF). Spontaneous production of MlP-1 was higher in the AA patients than the normal controls (1887±174 pg/ml vs 1643±93 pg/ml), but the difference was not significant. After LPS stimulation, the production of MIP-1 was markedly increased in the AA patients, and its level was significantly higher than that of the normal controls (2360±149 pg/ml vs 1517±92 pg/ml, p=0.0022). The level of TNF was also higher in the AA patients. However, IFN-, TGF-2, SCF, and IL-3 were not detectable in the PBMNC-CM of either AA patients or normals. The myelopoietic suppressing effect of AA-PBMNC-CM from each AA patient was significantly blocked by pretreatment with anti-TNF-, resulting in a colony-forming enhancement of 174%±12%. A similar effect was noted in six of 11 AA patients by pretreatment with anti-MIP-1. We conclude that TNF and MIP-1 can be overproduced by the PBMNC of some AA patients, which may play a role in the progression of AA.     

Serum interleukin-6, interleukin-8, andβ 2-Microglobulin in early assessment of severity of acute pancreatitis comparison with serum C-reactive protein

The aim of this study was to compare the sensitivity, specificity, and diagnostic accuracy of serum interleukin-6, interleukin-8, ₂-microglobulin, and C-reactive protein in the assessment of the severity of acute pancreatitis using commercial kits for their respective assays. Thirty-eight patients with acute pancreatitis (25 men, 13 women, mean age 59 years, range 16–97) were studied; the diagnosis was based on prolonged upper abdominal pain associated with a twofold increase of serum lipase, and it was confirmed by imaging techniques. According to the Atlanta criteria, 15 patients had severe illness and 23 had mild disease. The four serum markers were determined in all patients on admission, as well as daily for the following five days. On the first day of the disease, the sensitivity (calculated on patients with severe pancreatitis), specificity (calculated on patients with mild pancreatitis), and the diagnostic accuracy of these serum markers for establishing the severity of acute pancreatitis were 100%, 86%, and 91% for interleukin-6 (cutoff level 2.7 pg/ml); 100%, 81%, and 88% for interleukin-8 (cutoff level 30 pg/ml); 58%, 81%, and 73% for ₂-microglobulin (cutoff level 2.1 mg/liter); and 8%, 95%, and 64% for C-reactive protein (cutoff level 11 mg/dl). The results of our study indicate that, when assayed during the first 24 hr of disease onset, interleukin-6 and interleukin-8 are better markers than ₂-microglobulin or C-reactive protein for evaluating the severity of acute pancreatitis.     

Anti β2-glycoprotein I antibodies in a patient with catastrophic antiphospholipid syndrome

Summary Autoimmune antiphospholipid antibodies are a hallmark of patients with antiphospholipid syndrome, and require a protein cofactor, 2-glycoprotein I, to bind anionic phospholipids. In these same patients, moreover, IgG directly binding 2-glycoprotein I are described. We found high plasma titres of both IgM and IgG anti 2-glycoprotein I antibodies in a patient with catastrophic antiphospholipid syndrome. After passing plasma through a Sephacryl S-300 column, an identical distribution pattern between anti 2-glycoprotein I and anticardiolipin antibodies was observed. Moreover, when IgG immunocomplexes were isolated from a high molecular fraction, IgM anti-2-glycoprotein I and anticardiolipin antibodies were detected. Thus IgG, IgM and IgG-IgM complexes of anti-2-glycoprotein I antibodies are present at the same time in a patient with antiphospholipid syndrome.     

The β3-Adrenergic System and β3-Adrenergic Agonists

Reviews in Endocrine and Metabolic Disorders -